The downregulation of SASH1 expression promotes breast cancer occurrence and invasion accompanied by the activation of PI3K-Akt-mTOR signaling pathway

Abstract

SASH1 (SAM and SH3 domain containing 1) has been increasingly reported as a tumor suppressor gene. However, there is limited research on the role of SASH1 in breast cancer. This manuscript aims to investigate the mechanism of SASH1 in the occurrence, development, and prognosis of breast cancer. Firstly, we obtained RNA-sequencing data of the tumors from the Genomic Data Commons data portal website, along with the corresponding clinical information of patients. Pan-cancer analysis was performed to analyze the expression of SASH1 across all tumors. Univariate Cox regression analysis was used to assess the correlation between SASH1 expression and the prognosis of breast cancer patients. Then, immunohistochemistry was utilized to evaluate the expression levels of SASH1, p-Akt, p-PI3K, and p-mTOR in breast cancer tissue. Finally, a cell assay was employed to analyze the impact of SASH1 on the proliferation and invasion of breast cancer cells (MDA-MB-231). The results revealed that SASH1 expression is decreased in BRCA, LUSC, LUAD, CESC, ESCA, and COAD. Meta-analysis also found that SASH1 is downregulated in most tumor tissues, and the expression level of SASH1 in breast cancer was significantly lower than that in the control group (OR = 0.14, 95% CI = 0.08-0.25; P < 0.001). Further experimental validation showed that SASH1 expression is significantly downregulated in breast cancer tissue (38.33%, 23/60), and the overexpression of SASH1 can inhibit the proliferation and invasion of breast cancer cells accompanied by the suppression of PI3K-Akt-mTOR signaling pathway. Additionally, SASH1 overexpression can improve OS and RFS of breast cancer patients.

Keywords: Akt; Breast cancer; PI3K; Prognosis; SASH1; mTOR.

Fig. 1

The expression level of SASH1 gene in tumors and their corresponding normal tissues. bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cervical and endocervical cancer (CESC), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCA), head and neck squamous cell carcinoma (HNSC), kidney chromophobe (KICH), kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), sarcoma (SARC), skin cutaneous melanoma (SKCM), stomach adenocarcinoma (STAD), thyroid carcinoma (THCA), thymoma (THYM), uterine corpus endometrial carcinoma (UCEC).

Fig. 2

The flow diagram of study selection. CNKI, China National Knowledge Infrastructure.

Fig. 3

The relationship between the expression level of SASH1 and the risk of tumor. OR, odds ratio; CI confidence interval; SASH1, SAM—and SH3 domain containing 1.

Fig. 4

The rate of SASH1 mutations in breast cancer. (A), the mutation rate of SASH1 is relatively high in breast cancer. (B), the differential expression of SASH1 in breast cancer was observed in both breast cancer datasets. Patients were divided into a breast cancer group and a healthy control group. A linear model combined with the Empirical Bayes approach in the limma package was used to perform differential analysis between the two groups. (C), SASH1 has a high predictive value for the risk of breast cancer occurrence. SASH1, SAM—and SH3 domain containing 1.

Fig. 5

The expression levels of SASH1 are negatively correlated with p-Akt, p-PI3K, and p-mTOR in breast cancer tissues. (A, C), The expression levels of SASH1, p-Akt, p-PI3K, and p-mTOR in breast cancer tissues. (B), The correlation analysis between SASH1 and p-Akt, p-PI3K, and p-mTOR. SASH1, SAM—and SH3 domain containing 1; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; mTOR, mammalian target of rapamycin.

Fig. 6

The overexpression of SASH1 can inhibit the proliferation, migration, and invasion abilities of MDA-MB-231 cells. (A–D), The wound-closure assay and transwell invasion assay shown that SASH1 can inhibit the migration and invasion of MDA-MB-231 cells. (E), CCK8 assay shown that SASH1 can inhibit the proliferation of MDA-MB-231 cells. SASH1, SAM—and SH3 domain containing 1.

Fig. 7

The overexpression of SASH1 can inhibit the PI3K-Akt-mTOR signaling pathway. (A), Q-PCR showed successful construction of SASH1 overexpression cells (MDA-MB-231/pcDNA3.1-SASH1). (B), The expression levels of p-Akt, p-PI3K, and p-mTOR were significantly decreased in breast cancer cells of SASH1 overexpression (MDA-MB-231/pcDNA3.1-SASH1). SASH1, SAM—and SH3 domain containing 1; PI3K, phosphoinositide 3-kinase (This study explores PIK3 p110); Akt, protein kinase B (This study explores AKt-Thr308 phosphorylation sites); mTOR, mammalian target of rapamycin (This study explores mTORC1).

Fig. 8

The relationship between SASH1 expression levels and the prognosis of breast cancer patients. OS, overall survival; DMFS, distant metastasis free survival; RFS, recurrence free survival; and PPS, post-progression survival; SASH1, SAM—and SH3 domain containing 1.