Breast cancer is the most frequently diagnosed cancer and the second leading cause of cancer deaths in women worldwide. Clinically, this heterogeneous disease is categorized into three basic therapeutic groups: ER positive group, HER2 enriched group and triple-negative group. Triple-negative breast cancer (TNBC) lack of ER, PR and HER2 expression which generally occurs in younger women, is a group characterized by higher rates of relapse, greater metastatic potential, and shorter overall survival with only chemotherapy options. To date, the molecular mechanisms that drive TNBC occurrence have not been fully elucidated. Targeted therapies have not significantly improved survival in patients with TNBC [24, 35].
Previous work has mainly focused on genetic and transcriptional changes in TNBC. Proteolytic cleavage represents a unique and irreversible posttranslational event to regulate the function and half-life of many intracellular and extracellular proteins. ADAM10 belonging to the ADAM family of metalloproteinases which cleave and shed the ectodomain of hundreds of transmembrane proteins, plays important roles in physiological and pathophysiologcial processes. It has also been implicated in the pathogenesis of several types of human malignant tumors including breast cancer . But the roles of ADAM10 in breast cancer have been focused on the HER2 positive type [20–23] rather than on the triple-negative type [25, 37]. Here, we have demonstrated that the higher level of ADAM10 in its active form is expressed not only in HER2 amplified cell line SK-BR-3 but also in TNBC cell lines MDA-MB-231, MDA-MB-468 and BT-549, compared with ER positive cell line MCF7. Our findings also suggest that ADAM10 is biologically significant in TNBC. ADAM10 promotes migration, invasion, cell growth and cell-cycle progression, and blocks cell apoptosis of TNBC cells, with the activation of Notch signaling pathway.
Notch signaling system that contains four Notch receptors (Notch1- Notch4) and five canonical ligands(Dll1, Dll3, Dll4, Jagged1 and Jagged2), plays important roles including carcinogenesis, cancer stem cell renewal, angiogenesis, and chemotherapy resistance in the progression of breast cancer . Notch1 is highly expressed in poorly differentiated breast tumors and associated with poor overall survival . Specifically inhibiting human Notch1 in triple negative breast cancer enhances the antitumor efficacy of chemotherapy and decelerates tumor growth through reduction of cancer stem cells . Notch receptor is activated by ligand binding, followed by ADAM10/ADAM17 mediated proteolytic cleavage at Notch extracellular domain (NECD) site 2 (S2) [4, 31] and a second γ-secretase proteolytic cleavage at Notch transmembrane domain (NTM) site 3 (S3). These result in release of the Notch intracellular domain (NICD), which translocates to the nucleus and activates transcription of downstream target genes . But the regulation of Notch signaling by ADAM10 in TNBC has not been reported before. Our study has demonstrated that knockdown of ADAM10 using siRNA decreases the expression of Notch1. Meanwhile, the expression level of Notch1 targets Cyclin D3, HES1 and c-Myc is reduced and p21 Waf1/Cip1 is increased. Cyclin D3 regulates the cell cycle by controlling physiological progression from G1 to S phase , whereas the tumor suppressor p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression . HES1 and c-Myc have been implicated in cancer . Hence, we speculate that ADAM10 promotes cell cycle progression, growth and metastasis of TNBC cells by regulation of Notch1and its targets Cyclin D3, p21 Waf1/Cip1, Hes1 and c-Myc.
Due to lack of targeted therapies in TNBC, chemotherapy remains the standard of care. Chemotherapeutic drugs for breast cancer include epirubicin, doxorubicin, paclitaxel, and docetaxel, each aiming at blocking the proliferation of breast cancer cells. However, consequent chemo-resistance results in the failure of chemotherapy and tumor relapse. In our study, knockdown of ADAM10 could strengthen the sensitivity of chemotherapy drugs including paclitaxel and adriamycin in TNBC cells in vitro. It indicates that ADAM10 is associated with chemo-resistance in breast cancer especially in TNBC. But the underlying mechanism is still unknown.
The mechanisms of chemo-resistance in breast cancer include ATP-binding cassette (ABC) transporters such as P-gp transporting a variety of drugs outside the cell membrane, and formation of cancer stem cells (CSCs) which carry CD44 . We previously reported that PrPc promotes multidrug resistance in ER + breast cancer cell line MCF7/ADR by interacting with P-gp and CD44 . On the other hand, ADAM10 proteolytically cleaves and releases many important biologically active substrates including Notch receptors, PrPc  and CD44 . In HER2 + breast cancer, ADAM10 cleaves and sheds HER2 fragment p95HER2. Then anti-HER2 antibody such as trastuzumab cannot bind p95HER2 which has been proteolytically shed. So ADAM10 induces HER2 shedding and causes trastuzumab resistance . Based on these investigations, it is hypothesized that ADAM10 may be involved in the drug resistance of TNBC by regulating its substrates associated with chemo-resistance. In TNBC cells, we find that ADAM10 interacts with CD44 and PrPc and initiates their intramembrane proteolysis followed by nuclear transport and signaling of the cytoplasmic domain. These findings confirm our speculation that ADAM10 promotes drug resistance via regulating CD44 and PrPc in TNBC.
In clinical breast cancer patients receiving NACT, we divide them into two cohorts including Cohort 1 with poor response to NACT and Cohort 2 with good response to NACT, based on the effects of NACT. We found that the ADAM10 expression level of pre-NACT tissue samples is significantly higher in Cohort 1 than in Cohort 2. Inversely, after these patients received NACT, ADAM10 protein level of post-NACT samples is distinctly lower in Cohort 1 than in Cohort 2. Meanwhile, compared with the pre-NACT samples, the ADAM10 expression level of the post-NACT samples is decreased in Cohort 1 but increased in Cohort 2. We speculate that high ADAM10 protein level of pre-NACT samples accomponied by overexpression of its substrates such as PrPc and CD44 in Cohort 1 cases. High substrates level may impact ADAM10 activity by negative feedback regulation . Consequently, ADAM10 expression is decreased after NACT in Cohort 1 cases. These results suggest that high ADAM10 expression before NACT indicates poor chemotherapy effect. Using ADAM10 inhibitor in combination with chemotherapeutic drugs may reduce drug resistance and improve therapeutic efficacy. Furthermore, our data showed that high ADAM10 expression is an independent predictor of poor 5-year overall survival in breast cancer patients. However, due to the small number of TNBC cases we collected, the relationship between ADAM10 and the effect of neoadjuvant chemotherapy and overall survival in TNBC could not be determined.
Taking all data together, ADAM10 is involved in the oncogenic process of TNBC and may be provided as a biomarker and therapeutic target in TNBC. In fact, some ADAM10 inhibitors were tested in clinical trials including HER2 + breast cancer [47, 48], but ultimately failed and discontinued due to the ambiguous value. Nevertheless, there is no report on the application of ADAM10 inhibitors in TNBC. In our experiments, ADAM10 affects functions of TNBC by regulating its substrates Notch1 receptor, CD44 and PrPc. Most likely, it will be necessary to first analyze expression of ADAM10 substrates such as Notch receptors, CD44 and PrPc, when selecting patient populations for future ADAM10 inhibitors trials.