Notch1 promotes resistance to cisplatin by up-regulating Ecto-5'-nucleotidase (CD73) in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is an aggressive molecular subtype that due to lack of druggable targets is treated with chemotherapy as standard of care. However, TNBC is prone to chemoresistance and associates with poor survival. The aim of this study was to explore the molecular mechanisms of chemoresistance in TNBC. Firstly, we found that the mRNA expression of Notch1 and CD73 in cisplatin-treated patient material associated with poor clinical outcome. Further, both were upregulated at the protein level in cisplatin-resistant TNBC cell lines. Overexpression of Notch1 intracellular domain (termed N1ICD) increased expression of CD73, whereas knockdown of Notch1 decreased CD73 expression. Using chromatin immunoprecipitation and Dual-Luciferase assay it was identified that N1ICD directly bound the CD73 promoter and activated transcription. Taken together, these findings suggest CD73 as a direct downstream target of Notch1, providing an additional layer to the mechanisms underlying Notch1-mediated cisplatin resistance in TNBC.

Markers Associated With Tumor Recurrence in Patients With Breast Cancer Achieving a Pathologic Complete Response After Neoadjuvant Chemotherapy.

Background: Patients who achieve a tumor pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) have better outcomes than patients with residual tumor. However, tumors still recur in the pCR patients. Therefore, we aim to explore factors associated with tumor recurrence in this patient population. Methods: A total of 1,913 patients diagnosed with breast cancer between 1995 and 2020 and received NAC were included in this analysis. Clinicopathological data of the patients were retrospectively collected. We used Cox regression analysis to assess the associations of clinicopathological factors with patients' outcome. Proteomic study of tumors was applied to identify differentially expressed proteins (DEPs) between tumors from the pCR patients with tumor recurrence and tumors from those without tumor recurrence. PPI network analysis of the corresponding genes of DEPs was used to identify the hub genes. The prognostic value of the corresponding genes of DEPs was evaluated using two online databases, Kaplan-Meier Plotter and bc-GenExMiner. The genes that were significantly associated with patients' survival in both databases, as well as being identified as hub genes, were considered as potential prognostic markers for pCR patients. Publicly available data from Gene Expression Omnibus (GEO) was used to verify the prognostic value of the identified marker. Results: Among the 1,913 included patients, 420 had tumor pCR. The median follow-up for the pCR patients was 32.6 months (IQR, 16.3-55.5). Overall estimated 5-year risk of tumor recurrence for the pCR patients was 11%. Multivariable analysis showed that a higher pre-NAC clinical T stage and N stage were independent predictors for increased risk of tumor recurrence (hazard ratio [HR] 2.57, 95% confidence interval [CI] 1.01-6.51, P=0.047 for clinical T stage and HR 3.48, 95%CI 1.37-8.83, P=0.009 for clinical N stage). NAC regimens, the type of breast and axillary surgery, and adjuvant chemotherapy were not associated with tumor recurrence. Finally, aldehyde dehydrogenase (ALDH) 3A2 was identified by the proteomic study and was verified as a potential predictor for tumor recurrence in the pCR patients (with a median follow up of 3.78 years for dataset GSE32603 and 2.74 years for dataset GSE25066 from GEO, tumor recurrence rate: low versus high expression, 20.7% versus 4.5% [data from GSE32603]; 10.9% versus 0% [data from GSE25066]). Conclusions: Clinical T stage, clinical N stage and tumor expression of ALDH3A2 were potential markers for predicting tumor recurrence in the pCR patients after NAC.

CXCL9 Is a Potential Biomarker of Immune Infiltration Associated With Favorable Prognosis in ER-Negative Breast Cancer.

The chemokine CXCL9 (C-X-C motif chemokine ligand 9) has been reported to be required for antitumour immune responses following immune checkpoint blockade. In this study, we sought to investigate the potential value of CXCL9 according to immune responses in patients with breast cancer (BC). A variety of open-source databases and online tools were used to explore the expression features and prognostic significance of CXCL9 in BC and its correlation with immune-related biomarkers followed by subsequent verification with immunohistochemistry experiments. The CXCL9 mRNA level was found to be significantly higher in BC than in normal tissue and was associated with better survival outcomes in patients with ER-negative tumours. Moreover, CXCL9 is significantly correlated with immune cell infiltration and immune-related biomarkers, including CTLA4, GZMB, LAG3, PDCD1 and HAVCR2. Finally, we performed immunohistochemistry with breast cancer tissue samples and observed that CXCL9 is highly expressed in the ER-negative subgroup and positively correlated with the immune-related factors LAG3, PD1, PDL1 and CTLA4 to varying degrees. These findings suggest that CXCL9 is an underlying biomarker for predicting the status of immune infiltration in ER-negative breast cancer.

FABP7 is a potential biomarker to predict response to neoadjuvant chemotherapy for breast cancer.

BACKGROUND: Early prediction of response to neoadjuvant chemotherapy (NAC) is critical in choosing appropriate chemotherapeutic regimen for patients with locally advanced breast cancer. Herein, we sought to identify potential biomarkers to predict the response to neoadjuvant chemotherapy for breast cancer patients. METHODS: Three genomic profiles acquired by microarray analysis from subjects with or without residual tumors after NAC downloaded from the GEO database were used to screen the differentially expressed genes (DEGs). An array of public databases, including ONCOMINE, cBioportal, Breast Cancer Gene Expression Miner v4.0, and the Kaplan Meir-plotter, etc., were used to evaluate the potential functions, related signaling pathway, as well as prognostic values of FABP7 in breast cancer. Anti-cancer drug sensitivity assay, real-time PCR, flow cytometry and western-blotting assays were used to investigate the function of FABP7 in breast cancer cells and examine the relevant mechanism. RESULTS: Two differentially expressed genes, including FABP7 and ESR1, were identified to be potential indicators of response to anthracycline and taxanes for breast cancer. FABP7 was associated with better chemotherapeutic response, while ESR1 was associated with poorer chemotherapeutic effectiveness. Generally, the expression of FABP7 was significantly lower in breast cancer than normal tissue samples. FABP7 mainly high expressed in ER-negative breast tumor and might regulate cell cycle to enhance chemosensitivity. Moreover, elevated FABP7 expression increased the percentage of cells at both S and G2/M phase in MDA-MB-231-ADR cells, and decreased the percentage of cells at G0/G1 phase, as compared to control group. Western-blotting results showed that elevated FABP7 expression could increase Skp2 expression, while decrease Cdh1 and p27kip1 expression in MDA-MB-231-ADR cells. In addition, FABP7 was correlated to longer recurrence-free survival (RFS) in BC patients with ER-negative subtype of BC treated with chemotherapy. CONCLUSION: FABP7 is a potential favorable biomarker and predicts better response to NAC in breast cancer patients. Future study on the predictive value and detail molecular mechanisms of FABP7 in contribution to chemosensitivity in breast cancer is warranted.

O-6-methylguanine DNA methyltransferase is a favorable biomarker with proliferation suppressive potential in Breast Cancer.

Background: The O6-methylguanine-DNA methyltransferase (MGMT) is a highly effective enzyme capable of repairing DNA damage to maintain genomic stability. Until recently, reports on the expression and potential role of MGMT in breast cancer remain controversial. This study is intended to elucidate the prognostic significance and potential function of MGMT in breast cancer. Materials and methods: The immunohistochemistry assay and a series of public databases were utilized to determine the relevance between MGMT expression and clinicopathological characteristics, as well as survival outcomes in patients with breast cancer. The western blotting, qRT-PCR, proliferation, colony formation and transwell assays were used to investigate the potential function of MGMT in breast cancer cells. Results: The immunohistochemistry analysis and public cancer databases exploration demonstrated that MGMT expression was significantly related to estrogen receptor (ER) positivity in breast cancer. Positive expression of MGMT predicts a longer distant-free survival (DFS) and overall survival (OS) in patients with breast cancer, especially in ER-positive tumor. The mRNA level of MGMT was significantly associated with those of ESR1, GATA3 and FOXA1 in ER-positive breast tumor. Down-regulation of MGMT expression enhanced the proliferative and invasive capacities of breast cancer cells through PTEN/AKT pathway. Conclusions: MGMT is a favorable biomarker with proliferation suppressive potential in ER-positive breast cancer. Future study on targeted modulation of MGMT in the treatment of breast cancer is warranted.

Inhibition of Notch1 reverses EMT and chemoresistance to cisplatin via direct downregulation of MCAM in triple-negative breast cancer cells.

Resistance to chemotherapy continues to be a critical issue in the clinical therapy of triple-negative breast cancer (TNBC). Epithelial-mesenchymal transition (EMT) is thought to contribute to chemoresistance in several cancer types, including breast cancer. Identification of the key signaling pathway that regulates the EMT program and contributes to chemoresistance in TNBC will provide a novel strategy to overcome chemoresistance in this subtype of cancer. Herein, we demonstrate that Notch1 positively associates with melanoma cell adhesion molecule (MCAM), a unique EMT activator, in TNBC tissue samples both at mRNA and protein levels. High expression of Notch1 and MCAM both predicts a poor survival in basal-like/TNBC patients, particularly in those treated with chemotherapy. The expression of Notch1 and MCAM in MDA-MB-231 cells gradually increases in a time-dependent manner when exposing to low dose cisplatin. Moreover, the expressions of Notch1 and MCAM in cisplatin-resistant MDA-MB-231 cells are significantly higher than wild-type counterparts. Notch1 promotes EMT and chemoresistance, as well as invasion and proliferation of TNBC cells via direct activating MCAM promoter. Inhibition of Notch1 significantly downregulates MCAM expression, resulting in the reversion of EMT and chemoresistance to cisplatin in TNBC cells. Our study reveals the regulatory mechanism of the Notch1 pathway and MCAM in TNBC and suggesting that targeting the Notch1/MCAM axis, in conjunction with conventional chemotherapies, might be a potential avenue to enhance the therapeutic efficacy for patients with TNBC.

Upregulation of SOX11 enhances tamoxifen resistance and promotes epithelial-to-mesenchymal transition via slug in MCF-7 breast cancer cells.

Resistance to tamoxifen remains a prominent conundrum in the therapy of hormone-sensitive breast cancer. Also, the molecular underpinnings leading to tamoxifen resistance remain unclear. In the present study, we utilized the Gene Expression Omnibus database to identify that SOX11 might exert a pivotal function in conferring tamoxifen resistance of breast cancer. SOX11 was found to be markedly upregulated at both the messenger RNA and protein levels in established MCF-7-Tam-R cells compared to the parental counterparts. Moreover, SOX11 was able to activate the transcription of slug via binding to its promoter, resulting in promoting the progress of epithelial-to-mesenchymal transition and suppressing the expression of ESR1. Downregulating SOX11 expression can restore the sensitivity to 4-hydroxytamoxifen in MCF-7-Tam-R cells. Survival analysis from large sample datasets indicated that SOX11 was closely related to poorer survival in patients with breast cancer. These findings suggest a novel feature of SOX11 in contributing to tamoxifen resistance. Hence, targeting SOX11 could be a potential therapeutic strategy to tackle tamoxifen resistance in breast cancer.

GATA Binding Protein 3 Boosts Extracellular ATP Hydrolysis and Inhibits Metastasis of Breast Cancer by Up-regulating Ectonucleoside Triphosphate Diphosphohydrolase 3.

Despite remarkable advancements in our understanding of breast cancer, it remains the leading cause of cancer deaths in women. Distant recurrence and metastasis is the main reason for death due to breast cancer. It is well recognized that the GATA binding protein 3 (GATA3), a transcription factor, is a tumor suppressor in breast cancer. To date, the mechanistic molecular details of GATA3 remain elusive, because, as a transcription factor, it is not a direct executor in physiological and pathological processes. Here, we demonstrate that GATA3 reduces the ATP level in the breast cancer microenvironment and inhibits breast cancer metastasis by up-regulating ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3). The extracellular ATP concentration is significantly higher in tumor tissues than in normal tissues and promotes the migration of cancer cells from the primary site. ENTPD3 hydrolyzes ATP in tumor microenvironment and suppresses breast cancer metastasis. Furthermore, ENTPD3 inhibits epithelial-to-mesenchymal transition, a key program responsible for the development of metastatic disease. These findings provide novel insights into the tumor suppressor activity of GATA3.

Knockdown of nucleophosmin 1 suppresses proliferation of triple-negative breast cancer cells through activating CDH1/Skp2/p27kip1 pathway.

BACKGROUND: NPM1 is a multifunctional phosphoprotein that commutes between the cytoplasm and nucleus in cell cycle process, which appears to be actively involved in tumorigenesis. Herein, we sought to investigate the possible role and prognostic value of NPM1 in triple-negative breast cancer (TNBC). METHODS: An array of public databases, including bc-GenExMiner v4.0, GOBO, GEPIA, UAL-CAN, ONCOMINE database and Kaplan-Meier plotter, were used to investigate the expression feature and potential function of NPM1 in TNBC. Immunohistochemistry, immunofluorescence, proliferation and colony formation, flow cytometry and western-blotting assays were used to analyze and verify the function and relevant mechanism of NPM1 in TNBC tissues and cells. RESULTS: According to analysis from bc-GenExMiner, the expression level of NPM1 was significantly higher in basal-like subtypes than luminal-A, HER-2 or normal-like subtypes of breast cancer (P<0.0001). GOBO database analysis indicated that the expression of NPM1 in basal-A or basal-B was significantly higher than luminal-like breast cancer cells. Immunohistochemistry assay in 52 TNBC tissue samples showed that positive expression of Ki-67 was 93.5% in the high-NPM1-expression group and 66.7% in the low-NPM1-expression group, respectively (P=0.032). Proliferation and colony formation assays demonstrated that inhibition of NPM1 suppressed cell growth by approximately 2-fold and reduced the number of colonies by 3-4-fold in MDA-MB-231 and BT549 cells. Moreover, inhibition of NPM1 in MDA-MB-231 and BT549 cells increased the percentage of cells at G0/G1 phase and decreased the percentage of cells at both S and G2/M phase, as compared with control counterparts. Western-blotting results showed that down-regulation of NPM1 could elevate CDH1 and p27kip1 expression, while decrease Skp2 expression both in MDA-MB-231 and BT549 cells. In addition, high mRNA expression of NPM1 correlated with shorter RFS (HR=1.64, P=0.00013) and OS (HR=2.45, P=0.00034) in patients with TNBC. CONCLUSIONS: NPM1 is significantly high expressed basal-like/triple-negative breast cancer and is correlated with shorter RFS and OS in this subset of patients. Knockdown of NPM1 impairs the proliferative capacity of TNBC cells via activation of the CDH1/Skp2/p27kip1 pathway. Targeting NPM1 is a potential therapeutic strategy against TNBC.

Major vault protein is a direct target of Notch1 signaling and contributes to chemoresistance in triple-negative breast cancer cells.

Resistance to chemotherapy remains a significant problem in the treatment of breast cancer, especially for triple-negative breast cancer (TNBC), in which standard systemic therapy is currently limited to chemotherapeutic agents. Our study aimed to better understand the molecular mechanisms that lead to failure of chemotherapy in TNBC. Herein, we observed elevated expression of Notch1 and major vault protein (MVP) in MDA-MB-231DDPR cells compared to their parental counterparts. We demonstrated that Notch1 could positively regulate the expression of MVP. Also, Notch1 intracellular domain (ICD) was capable of binding to CBF-1 on the promoter of MVP to drive its transcription, resulting in activation of AKT pathway and promoting the progress of epithelial to mesenchymal transition (EMT). Conversely, silencing of Notch1 and MVP suppressed AKT pathway, reduced EMT and enhanced the sensitivity of TNBC cells to cisplatin and doxorubicin. Survival analysis indicated that the MVP was closely related to shorter recurrence-free survival (RFS) in patients with TNBC. Collectively, this study provides evidence that Notch1 activates AKT pathway and promotes EMT partly through direct activation of MVP. Targeting Notch1/MVP pathway appears to have potential in overcoming chemoresistance in TNBC.

MiR-221/222 promote epithelial-mesenchymal transition by targeting Notch3 in breast cancer cell lines.

Basal-like breast cancer (BLBC) is an aggressive subtype with a strong tendency to metastasize. Due to the lack of effective chemotherapy, BLBC has a poor prognosis compared with luminal subtype breast cancer. MicroRNA-221 and -222 (miR-221/222) are overexpressed in BLBC and associate with metastasis as well as poor prognosis; however, the mechanisms by which miR-221/222 function as oncomiRs remain unknown. Here, we report that miR-221/222 expression is inversely correlated with Notch3 expression in breast cancer cell lines. Notch3 is known to be overexpressed in luminal breast cancer cells and inhibits epithelial to mesenchymal transition (EMT). We demonstrate that miR-221/222 target Notch3 by binding to its 3' untranslated region and suppressing protein translation. Ectopic expression of miR-221/222 significantly promotes EMT, whereas overexpression of Notch3 intracellular domain attenuates the oncogenic function of miR-221/222, suggesting that miR-221/222 exerts its oncogenic role by negatively regulating Notch3. Taken together, our results elucidated that miR-221/222 promote EMT via targeting Notch3 in breast cancer cell lines suggesting that miR-221/222 can serve as a potential therapeutic target in BLBC.

MCAM/CD146 promotes tamoxifen resistance in breast cancer cells through induction of epithelial-mesenchymal transition, decreased ERα expression and AKT activation.

Tamoxifen resistance presents a prominent clinical challenge in endocrine therapy for hormone sensitive breast cancer. However, the underlying mechanisms that contribute to tamoxifen resistance are not fully understood. In this study, we established a tamoxifen resistant MCF-7 cell line (MCF-7-Tam-R) by continuously incubating MCF-7 cells with 4-OH-tamoxifen. We found that melanoma cell adhesion molecule (MCAM/CD146), a unique epithelial-to-mesenchymal transition (EMT) inducer, was significantly up-regulated at both mRNA and protein levels in MCF-7-Tam-R cells compared to parental MCF-7 cells. Mechanistic research demonstrated that MCAM promotes tamoxifen resistance by transcriptionally suppressing ERα expression and activating the AKT pathway, followed by induction of EMT. Elevated MCAM expression was inversely correlated with recurrence-free and distant metastasis-free survival in a cohort of 4142 patients with breast cancer derived from a public database, particularly in the subgroup only treated with tamoxifen. These results demonstrate a novel function of MCAM in conferring tamoxifen resistance in breast cancer. Targeting MCAM might be a promising therapeutic strategy to overcome tamoxifen resistance in breast cancer patients.