circSLC8A1 sponges miR-671 to regulate breast cancer tumorigenesis via PTEN/PI3k/Akt pathway.

Breast cancer is the most frequently diagnosed and the leading cause of cancer-related deaths in women worldwide. However, the role of circSLC8A1 in breast cancer remains elusive. Herein, a cohort of 77 breast tumors and paired adjacent normal mammary tissues were collected. We demonstrated that circSLC8A1 was significantly down-regulated in breast cancer tissues and cell lines, of which expression was negatively correlated with clinical severity and dismal prognosis. Overexpression of circSLC8A1 suppressed cell proliferation, migration and invasion in vitro, and inhibited tumor growth in vivo. CircSLC8A1 directly targeted miR-671 to execute tumor suppressive activities via regulating PI3k/Akt signaling. Krüppel-like factor 16 (KLF16), a transcriptional activator of PTEN, was identified as a target of miR-671. Furthermore, circSLC8A1 could sponge miR-671 to suppress breast tumor growth via PTEN/PI3k/Akt signaling in vivo. In summary, circSLC8A1/miR-671 regulates breast cancer progression through PTEN/PI3k/Akt signaling, which may provide efficient therapeutic target for this devastating cancer.

Long non-coding RNA MIR100HG promotes the migration, invasion and proliferation of triple-negative breast cancer cells by targeting the miR-5590-3p/OTX1 axis.

BACKGROUND: As an aggressive subtype of breast cancer with a high risk of recurrence, triple-negative breast cancer (TNBC) lacks available treatment targets. LncRNA MIR100HG promotes cell proliferation in TNBC. However, few studies have investigated the molecular mechanism of MIR100HG in TNBC. Thus, additional in-depth investigations are needed to unravel its associated regulatory mechanism. METHODS: MIR100HG and miR-5590-3p expression in TNBC tissue samples and cell lines was detected by RT-qPCR. Flow cytometry, transwell, wound-healing, CCK8 and colony formation assays were performed to analyse cell apoptosis, cell cycle, invasion, migration and proliferation. The protein expression of orthodenticle homeobox 1 (OTX1) and proteins in the ERK/MAPK signalling pathway were assessed by western blot analysis. Bioinformatics and luciferase assay were performed to predict and validate the interaction between MIR100HG and miR-5590-3p as well as OTX1 and miR-5590-3p. RNA immunoprecipitation (RIP) was used to detect the interaction between MIR100HG and miR-5590-3p. Subcutaneous tumour growth was observed in nude mice. Immunohistochemistry (IHC) analysis was used to assess OTX1 expression in tumour tissues. RESULTS: MIR100HG expression was upregulated, whereas that of miR-5590-3p was downregulated in TNBC. MIR100HG was shown to directly interact with miR-5590-3p. Furthermore, MIR100HG knockdown could promote TNBC cell apoptosis and cell cycle arrest in G0/G1 phase while inhibiting migration, invasion and proliferation. Furthermore, miR-5590-3p inhibition showed the opposite results and could reverse the effect of MIR100HG knockdown in TNBC cells. MiR-5590-3p downregulated the ERK/MAPK signalling pathway, suppressed the migration, invasion and proliferation of TNBC cells and promoted their apoptosis and cell cycle arrest in G0/G1 phase by targeting OTX1. In addition, MIR100HG knockdown inhibited OTX1 expression by upregulating miR-5590-3p in vivo, thereby inhibiting tumour growth. CONCLUSIONS: MIR100HG promotes the progression of TNBC by sponging miR-5590-3p, thereby upregulating OTX1, suggesting a new potential treatment target for TNBC.

Relative dose intensity and therapy efficacy in different breast cancer molecular subtypes: a retrospective study of early stage breast cancer patients treated with neoadjuvant chemotherapy.

To investigate the relationship between chemotherapy dose intensity and therapy efficacy of different molecular subtypes. Clinical and pathological features of the patients with breast cancer were retreived from the hospital records. 315 patients were analyzed (251 showed clinical response, 38 acquired pCR). Patients with positive ER status, negative PR status, higher Ki67 level and higher RTDI had better therapy response. 13.5 and 84.5 % were identified the benchmark of Ki67 and RTDI, respectively. As the result of interior-subgroup comparison, luminal subgroups acquired better response rate when RTDI ≥ 84.5 %. In patients of luminal breast cancer, tumor size change arose from increasing of dose intensity and finally showed reached a plateau after RTDI ≥ 95 % (r (2) = 0.303, p < 0.001). As the result of intersubgroup comparison, TNBC patients were more likely to acquired better clinical and pathology response when RDTI < 84.5 %. Ki67 change arose sharply from increasing of dose intensity when RDTI < 84.5 % (r (2) = 0.656, p < 0.001), whereas the regression curve showed a terminal plateau in patients of RDTI ≥ 84.5 % (r (2) = 0.427, p < 0.001). Given lower RTDI, luminal patients are less likely to achieve response, and TNBC patients are associated with higher response rate. Dissimilar of therapy efficacy between luminal subtype and TNBC becomes inconspicuous as RTDI rises. Chemosensitivity may associate with dose intensity, especially in luminal subtypes, and tailored therapeutic strategies should be considered.

LncRNA TYMSOS facilitates breast cancer metastasis and immune escape through downregulating ULBP3.

The focus of the study is to examine the function of TYMSOS in immune escape of breast cancer, which is the most frequently diagnosed malignancy among women globally. Our study demonstrated that upregulated TYMSOS was associated with unfavorable prognosis and immune escape in breast cancer. TYMSOS promoted the malignant phenotypes of breast cancer cells, and reduced the cytotoxicity of NK92 cells on these cells. CBX3 was a downstream effector in TYMSOS-induced malignant phenotypes in breast cancer cells. Mechanistic studies showed that TYMSOS facilitated CBX3-mediated transcriptional repression of ULBP3, and it also promoted SYVN1-mediated ubiquitin-proteasomal degradation of ULBP3. TYMSOS promoted cell growth, metastasis, and immune escape via CBX3/ULBP3 or SYVN1/ULBP3 axis. The in vivo studies further showed that silencing of TYMSOS repressed tumor growth and boosted NK cell cytotoxicity. In sum, TYMSOS boosted breast cancer metastasis and immune escape via CBX3/ULBP3 or SYVN1/ULBP3 axis.

YAP1/MMP7/CXCL16 axis affects efficacy of neoadjuvant chemotherapy via tumor environment immunosuppression in triple-negative breast cancer.

BACKGROUND: To evaluate the association of potential YAP1/MMP7/CXCL16 axis and tumor infiltrating lymphocytes (TILs) related chemo-response in triple-negative breast cancer (TNBC) patients. METHODS: We estimated the messenger RNA (mRNA) expression levels of Yes-associated protein 1 (YAP1), MMP7, and CXCL16 in paired TNBC tumor/para-tumor tissues by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and performed statistical analysis according to neoadjuvant chemotherapy (NAC) response. Based on The Cancer Genome Atlas (TCGA) data, we noticed outstanding expression of MMP7/CXCL16 in TNBC cases, as well as associations between MMP7/CXCL16 and HIPPO-YAP1-relevant kinases. We also performed gene set enrichment analysis (GSEA) between MMP7/CXCL16 and YAP1-associated pathways. Western blotting assay was employed to evaluate YAP1/MMP7/CXCL16 expression in vitro and their modulation sequence. Logistic model stepwise regression analysis was used to assess YAP1, MMP7, CXCL16, and TILs as therapeutic predictors. Residual cancer burden (RCB) score was calculated and statistically analyzed according to intensity of these variables, and receiver operating characteristic (ROC) curve also showed their predictive value in NAC response. Recruitment efficacy for CD4+/CD8+ TIL cells (TCGA data) as well as quantified TIL cells density were both explored according to YAP1, MMP7, and CXCL16 expression level. RESULTS: Up-regulation of YAP1/MMP7 and down-regulation of CXCL16 were both significant in TNBC cases with poor NAC response. Inhibition of YAP1 induced down-regulation of MMP7 and up-regulation of CXCL16, whereas inhibition of MMP7 also induced up-regulation of CXCL16. It was also shown that MMP7/CXCL16 was enriched in the YAP1-related pathway. Activation of the YAP1/MMP7/CXCL16 axis obviously affected RCB of TNBC cases. The ROC curve also supported the predictive value of YAP1/MMP7/CXCL16 axis and TILs density in NAC response prospect. The density of TILs, meanwhile, demonstrated a strong link with the YAP1/MMP7/CXCL16 axis. Over expression of YAP1/MMP7 significantly suppressed recruitment of CD4+/CD8+ TILs, while CXCL16 over expression had a beneficial impact on anti-tumor immune. CONCLUSIONS: Over expression of causes up-regulation of MMP7 and down-regulation of CXCL16, which suppressed CD4+/CD8+ TILs recruitment and indirectly affected NAC response of TNBC patients.

lncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis.

Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer. This study investigated the molecular mechanism and influences of MIR503HG, miR-224-5p, and homeobox A9 (HOXA9) on TNBC cell growth and migration. Dual-luciferase reporter gene and RNA immunoprecipitation were performed to examine the regulation of MIR503HG, miR-224-5p, and HOXA9. Cell proliferation, apoptosis, migration, and invasion were evaluated by colony formation, flow cytometry, and Transwell assays. Finally, nude mice were employed to investigate the influence of MIR503HG on TNBC tumor growth. HOXA9 protein levels were detected by immunohistochemical staining. MIR503HG and HOXA9 expression were reduced in TNBC, while miR-224-5p was increased. Overexpression of MIR503HG or HOXA9 reduced the cell migration ability and proliferation and promoted apoptosis, and knockdown of MIR503HG or overexpression of miR-224-5p exhibited the opposite effects. Furthermore, MIR503HG promoted HOXA9 expression by inhibiting miR-224-5p. Overexpression of miR-224-5p reversed the effects of MIR503HG overexpression on TNBC cells, while overexpression of HOXA9 reversed the effect of MIR503HG knockdown. Additionally, an in vivo study proved that MIR503HG inhibited TNBC tumor growth via the miR-224-5p/HOXA9 axis. MIR503HG inhibited cell proliferation and promoted the apoptosis of TNBC cells via the miR-224-5p/HOXA9 axis, which may function as a novel target for the treatment of TNBC.

The long non-coding RNA DANCR regulates the inflammatory phenotype of breast cancer cells and promotes breast cancer progression via EZH2-dependent suppression of SOCS3 transcription.

Long non-coding RNA (lncRNA) is involved in the regulation of tumorigenesis and metastasis. In this study, we focused on the clinical relevance, biological effects, and molecular mechanisms of the lncRNA differentiation antagonizing non-protein coding RNA (DANCR) in breast cancer. We compared the expression of DANCR between breast cancer and normal tissues, and between breast cancer cell lines and normal breast epithelial cells using quantitative real-time PCR (qRT-PCR) analysis. By knocking down and overexpressing DANCR, we assessed its significance in regulating viability (MTT assay), migration/invasion (Transwell assay), epithelial-mesenchymal transition (western blot), stemness (mammosphere formation assay and western blot), and production of inflammatory cytokines (qRT-PCR and ELISA) of breast cancer cells in vitro, as well as xenograft growth in vivo. Furthermore, using ChIP and RNA immunoprecipitation, we examined the reciprocal regulation between DANCR and suppressor of cytokine signaling 3 (SOCS3) in breast cancer. DANCR was significantly up-regulated in tissue samples from patients with breast cancer, as well as in breast cancer cell lines, as compared with normal tissues and breast epithelial cells, respectively. The highest DANCR expression levels were associated with advanced tumor grades or lymph node metastasis. DANCR was necessary and sufficient to control multiple malignant phenotypes of breast cancer cells in vitro and xenograft growth in vivo. Mechanistically, DANCR promoted the binding of enhancer of zeste homolog 2 (EZH2) to the promoter of SOCS3, thereby epigenetically inhibiting SOCS3 expression. Functionally, SOCS3 up-regulation or EZH2 inhibition could rescue multiple malignant phenotypes induced by DANCR. Our data indicate that DANCR is a pleiotropic oncogenic lncRNA in breast cancer. Boosting SOCS3 expression may reverse the oncogenic activities of DANCR and thus provide a therapeutic strategy for breast cancer treatment.

LncRNA GNAS-AS1 facilitates ER+ breast cancer cells progression by promoting M2 macrophage polarization via regulating miR-433-3p/GATA3 axis.

OBJECTIVE: ER+ breast cancer is the most common type of breast cancer, which seriously affects the physical and mental health of women. Recently, lncRNAs mediated tumor-associated macrophages (TAM) were identified to involve in tumorigenesis. Therefore, the present study aimed at demonstrating the regulatory network of GNAS-AS1 in TAM-mediated ER+ breast cancer progress. METHODS: The expression levels of genes were evaluated using qRT-PCR. The proportions of polarized macrophages (M1, M2) were assessed by flow cytometry. Cell proliferation, migration and invasion were evaluated by CCK-8, wound healing and transwell assay, respectively. Double-luciferase reporter system was used to detect the interaction between molecules. Western blot was applied to test protein levels. RESULTS: The expression of GNAS-AS1 was obviously increased in ER+ breast cancer tissues and cell lines, as well as M2 macrophages. GNAS-AS1 facilitated the capabilities of proliferation, migration and invasion of ER+ breast cancer cells by accelerating M2 macrophage polarization via directly sponging miR-433-3p. GATA3, as a target of miR-433-3p, could positively regulate by GNAS-AS1. Furthermore, either miR-433-3p overexpression or GATA3 knockdown impaired the effects of GNAS-AS1 on M2 macrophage polarization and ER+ breast cancer cells progression. CONCLUSION: GNAS-AS1/miR-433-3p/GATA3 axis promoted proliferation, metastasis of ER+ breast cancer cells by accelerating M2 macrophage polarization. The mechanism may provide a new strategy and target for ER+ breast cancer treatment.

miR­574­5p attenuates proliferation, migration and EMT in triple­negative breast cancer cells by targeting BCL11A and SOX2 to inhibit the SKIL/TAZ/CTGF axis.

Triple­negative breast cancer (TNBC) is a subtype of breast cancer with a high degree of malignancy. TNBC is prone to distant metastasis and has a poor prognosis. A number of TNBC­related microRNAs (miRNAs) have been studied and identified. However, the detailed roles of miR­574­5p in TNBC remain poorly understood. miR­574­5p, SRY (sex determining region Y)­box 2 (SOX2), B­cell lymphoma/leukaemia 11A (BCL11A), SKI like proto­oncogene (SKIL) and epithelial­mesenchymal transition (EMT)­related miRNAs and proteins were measured by reverse transcription­quantitative PCR and western blotting analysis, respectively. A luciferase reporter assay was employed to validate the direct targeting of SOX2 and BCL11A by miR­574­5p. MTT, colony formation and Transwell assays were performed to analyse the biological functions of miR­574­5p in TNBC cells. A nude mouse xenograft model was used to verify the effects of miR­574­5p on the tumorigenesis of TNBC in vivo. The results demonstrated that miR­574­5p levels were decreased in breast cancer tissues and cells. miR­574­5p repressed proliferation, migration and EMT in TNBC cells. Further experiments confirmed that miR­574­5p reduced tumour size and metastasis in vivo. miR­574­5p targeted BCL11A and SOX2 to inhibit the SKIL/transcriptional co­activator with PDZ­binding motif/connective tissue growth factor axis, and the inhibitory effect of miR­574­5p in TNBC cells was at least partly dependent on SOX2 and BCL11A. In addition, the regulation of downstream oncogenes by SOX2 was dependent on BCL11A. To the best of our knowledge, this is the first study to report the association between the miR­574­5p/BCL11A/SOX2 axis and the tumorigenesis of TNBC, which provides a new mechanism for understanding the progression of TNBC.

Clinical significance of androgen receptor expression in triple negative breast cancer-an immunohistochemistry study.

Androgen receptor (AR) is closely associated with the occurrence and progression of breast cancer; however, the clinical significance of it in triple negative breast cancer (TNBC) has been controversial. There is a limited amount of research regarding the effect of neoadjuvant chemotherapy on AR expression. By examining the expression of AR in patients with TNBC, the aim of the present study is to explore the clinical significance of AR and provide evidence for AR-directed treatment in TNBC. A total of 188 patients with primary TNBC with complete medical records were included in this retrospective study. Tumor sections from 41 patients (21.8%) were positive for AR, which was more often detected in small tumors (P=0.042) and cases with no lymph node involvement (P=0.032). Among them, 102 were treated with neoadjuvant chemotherapy (NAC). A total of 17 patients (16.7%) exhibited pathological complete response. However, the patient response was irrelevant to AR expression. Matched pathological tissues before and after NAC were collected for 49 cases, suggesting an enrichment of AR-expressing tumors following chemotherapy (P=0.008). Further analysis indicated that AR expression had no correlation with the disease-free and overall survival of patients with general TNBC; rather, it predicted a poor survival of the patients with stage III TNBC in comparison with those at earlier stages (P=0.035). AR expression occurs more often in small TNBC tumors or in cases with no lymph node metastasis. It is associated with a poor prognosis of the patients with advanced stages of tumors.

Second intron of mouse nestin gene directs its expression in pluripotent embryonic carcinoma cells through POU factor binding site.

Nestin, an intermediate filament protein, is expressed in the neural stem cells of the developing central nervous system. This tissue-specific expression is driven by the neural stem cell-specific enhancer in the second intron of the nestin gene. In this study, we showed that the mouse nestin gene was expressed in pluripotent embryonic carcinoma (EC) P19 and F9 cells, not in the differentiated cell types. This cell type-specific expression was conferred by the enhancer in the second intron. Mutation of the conserved POU factor-binding site in the enhancer abolished the reporter gene expression in EC cells. Oct4, a Class V POU factor, was found to be coexpressed with nestin in EC cells. Electrophoretic mobility-shift assays and supershift assays showed that a unique protein-DNA complex was formed specifically with nuclear extracts of EC cells, and Oct4 protein was included. Together, these results suggest the functional relevance between the conserved POU factor-binding site and the expression of the nestin gene in pluripotent EC cells.