Negative Breast Cancer Cell Proliferation Research Articles

MicroRNA-496 inhibits triple negative breast cancer cell proliferation by targeting Del-1.

Del-1 has been linked to the pathogenesis of various cancers, including breast cancer. However, the regulation of Del-1 expression remains unclear. We previously reported the interaction between microRNA-137 (miR-137) and the Del-1 gene. In this study, we investigated miR-496 and miR-137 as regulators of Del-1 expression in triple negative breast cancer (TNBC). Del-1 mRNA and miR-496 were measured by quantitative PCR in breast cancer cells (MDA-MB-231, MCF7, SK-BR3, and T-47D) and tissues from 30 patients with TNBC. The effects of miR-496 on cell proliferation, migration, and invasion were determined with MTT, wound healing, and Matrigel transwell assays, respectively. In MDA-MB-231 cells, miR-496 levels were remarkably low and Del-1 mRNA levels were higher than in other breast cancer cell lines. Luciferase reporter assays revealed that miR-496 binds the 3′-UTR of Del-1 and Del-1 expression is downregulated by miR-496 mimics. Furthermore, miR-496 inhibited the proliferation, migration, and invasion of MDA-MB-231 cells. The effects of miR-496 on cell proliferation were additive with those of miR-137, another miRNA that regulates Del-1 expression. Moreover, in the 30 TNBC specimens, miR-496 was downregulated (P < .005) and the levels of Del-1 in the plasma were significantly elevated as compared with in normal controls (P = .0142). The Cancer Genome Atlas (TCGA) data showed the correlation of miR-496 expression with better overall survival in patients with early TNBC. In in silico and in vitro analyses, we showed that Del-1 is a target of miR-496 in TNBC and thereby affects cancer progression. Our findings suggest that miR-496 and miR-137 additively target Del-1 and act as modulating factors in TNBC. They are potentially new biomarkers for patients with TNBC.

Anti-progranulin/GP88 antibody AG01 inhibits triple negative breast cancer cell proliferation and migration.

Triple negative breast cancer (TNBC) is characterized by invasiveness and short survival. Identifying novel TNBC-targeted therapies, to potentiate standard of care (SOC) therapy, is an unmet need. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in several cancers including breast cancer (BC). PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes. Since PGRN/GP88 expression is elevated in 30% TNBC, we investigated the involvement of progranulin on TNBC. The effect of inhibiting PGRN/GP88 expression in TNBC cells by siRNA was investigated. The effects of a neutralizing anti-human PGRN/GP88 monoclonal antibody AG01 on the proliferation and migration of two TNBC cell lines expressing PGRN/GP88 were then examined in vitro and in vivo. Inhibition of GP88 expression by siRNA and AG01 treatment to block PGRN/GP88 action reduced proliferation and migration in a dose-dependent fashion in MDA-MB-231 and HS578-T cells. Western blot analysis showed decreased expression of phosphorylated protein kinases p-Src, p-AKT, and p-ERK upon AG01 treatment, as well as inhibition of tumor growth and Ki67 expression in vivo. PGRN/GP88 represents a therapeutic target with companion diagnostics. Blocking PGRN/GP88 with antibody treatment mayprovide novel-targeted solutions in TNBC treatment which could eventually address the issue of toxicity and unresponsiveness associated with SOC.

MicroRNA-205-5p targets the HOXD9-Snail1 axis to inhibit triple negative breast cancer cell proliferation and chemoresistance.

MicroRNA-205 (miR-205) is believed to be related to the progress of tumors. HOXD9 has been proved to be expressed abnormally in several kinds of cancers. However, the role of miR-205 and HOXD9 in breast cancer remains unclear. The biological role of miR-205 in breast cancer cell proliferation and chemoresistance was investigated. The expression of miR-205 in clinical tissues and breast cancer cell lines were analyzed using quantitative real-time PCR test (qRT-PCR). Overexpression and knockdown models of miR-205 were established to study cell proliferation and chemotherapy-resistant. Moreover, the potential relationships between miR-205 and HOXD9/Snail1 were measured using qRT-PCR, western blot, and chemotherapy-resistant study. miR-205 was lowly expressed in breast cancer tissues and cell lines. Overexpression of miR-205 could inhibit cell proliferation and chemotherapy-resistance. Moreover, we proved that miR-205 could target the HOXD9-Snail1 axis to suppress triple negative breast cancer cell proliferation and chemoresistance. The activation of Snail1 gene by HOXD9 was also proved in this study. The present study may provide a novel insight for the therapeutic strategies of breast cancer through targeting miR-205/HOXD9/Snail1.

Long non-coding RNA LRRC75A-AS1 facilitates triple negative breast cancer cell proliferation and invasion via functioning as a ceRNA to modulate BAALC

As a common female malignancy, triple-negative breast cancer (TNBC) is the most serious subtype in breast cancer (BC). BAALC binder of MAP3K1 and KLF4 (BAALC) is a common oncogene in acute myelocytic leukemia (AML). We sought to explore the role of BAALC in TNBC. In this study, BAALC was significantly upregulated in TNBC tissues and cells. Then, the results of functional assays disclosed that BAALC facilitated cell proliferation, invasion, and epithelial–mesenchymal transition (EMT) processes, but repressed cell apoptosis in TNBC. Next, miR-380–3p was identified as the upstream of BAALC in TNBC cells. Moreover, LRRC75A-AS1 (also named small nucleolar RNA host gene 29: SNHG29) was verified to act as the sponge of miR-380–3p to elevate BAALC expression in TNBC. Besides, LRRC75A-AS1 could negatively regulate miR-380–3p but positively regulate BAALC expression. Finally, rescue assays elucidated that LRRC75A-AS1 facilitated cell proliferation, invasion, and EMT processes in TNBC by targeting miR-380–3p/BAALC pathway. Taken together, our study revealed a novel ceRNA network of LRRC75A-AS1/miR-380–3p/BAALC in accelerating TNBC development, indicating new promising targets for TNBC treatment.

LncRNA AFAP1-AS1 promotes triple negative breast cancer cell proliferation and invasion via targeting miR-145 to regulate MTH1 expression

The actin fiber-associated protein 1-antisense RNA1 (AFAP1-AS1) is upregulated in various cancers and associated with cancer proliferation and metastasis. Several cancer-related pathways have been linked to up-expression of this long non-coding (lnc)RNA, but the underlying mechanisms are yet unknown. In triple negative breast cancer (TNBC), AFAP1-AS1 expression is also significantly overexpressed compared to that in other subtypes of breast cancer from the TCGA dataset. In this study, we performed bioinformatic RNAhybrid analyses and identified that miR-145 is a potential target of AFAP1-AS1 and able to reduce MutT homolog-1 (MTH1) expression. Thus, this study investigated the oncogenic activity of AFAP1-AS1 in TNBC cells and the underlying mechanisms that are yet poorly understood. The results showed that miR-145 expression was low, whereas AFAP1-AS1 and MTH1 expression was high in TNBC cells and that miR-145 mimics reduced TNBC cell proliferation and invasion, whereas miR-145 knockdown exerted the opposite activity in TNBC cells. Moreover, knockdown of AFAP1-AS1 reduced tumor cell proliferation and invasion, but miR-145 co-transfection rescued tumor cell viability and colony formation ability. The dual luciferase reporter assay showed that AFAP1-AS1 could directly target miR-145, while miR-145 could directly target MTH1. After knockdown of ATF6, AFAP1-AS1 was reduced along with AFAP1-AS1 promoter activity. This study revealed that AFAP1-AS1 could promote TNBC cell proliferation and invasion via regulation of MTH1 expression through targeting of miR-145.

Abstract 3547: MicroRNA-496 inhibits triple negative breast cancer cell proliferation by targeting DEL-1

Abstract Background and Objectives: Del-1 is linked to the pathogenesis of various cancers including breast cancer; however, the regulation of Del-1 expression remains unclear. The current study investigated how microRNA-496 (miR-496) regulates Del-1 expression in triple negative breast cancer (TNBC). Methods: Del-1 mRNA and miR-496 were measured by quantitative PCR in breast cancer cells (MDA-MB-231, MCF7, SK-BR3, and T-47D) and tissues from 30 patients with TNBC. The effects of miR-496 on cell proliferation, migration, and invasion were determined in MTT, wound healing, and Matrigel Transwell assays, respectively. Results: In MDA-MB-231, miR-496 levels were remarkably low and Del-1 mRNA was higher compared to other breast cancer cell lines. Luciferase reporter assays revealed that miR-496 binds the 3′-UTR of Del-1 and that Del-1 expression is downregulated by miR-496 mimics. Furthermore, miR-496 inhibited the proliferation, migration, and invasion of MDA-MB-231 cells. The effects of miR-496 on cell proliferation were additive with those of miR-137, another miRNA that regulates Del-1 expression. Moreover, in the 30 TNBC specimens, miR-496 was downregulated (P &amp;lt; 0.005) and the levels of Del-1 in the plasma was significantly elevated as compared to normal controls (P = 0.0142). TCGA data showed the correlation of miR-496 expression with better overall survival in patients with early TNBC. Conclusions: In in silico and in vitro analyses, we showed that Del-1 is a target of miR-496 in TNBC and thereby affects cancer progression. Our findings suggest that miR-496 and Del-1 might act as modulating factors in TNBC and are new biomarkers for patients with TNBC. Citation Format: Dong Won Baek, Jae-Hwan Jeong, Soo Jung Lee, Jiyeon Lee, Yee Soo Chae, Wan WooK Kim, Jieun Kang, Ho Yong Park, Jin Hyang Jung, Ji Yun Jeong, Ji Young Park, Keon Uk Park. MicroRNA-496 inhibits triple negative breast cancer cell proliferation by targeting DEL-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3547.

Abstract P5-05-12: Progesterone drives ER-positive and triple negative breast cancer cell proliferation through progesterone receptor membrane component 1

Abstract Introduction: The role of progesterone and its receptors in breast cancer progression continue to be studied but remain controversial. Progesterone membrane receptors with the ability to regulate kinase signals, mediating breast cancer proliferation have been demonstrated. Increased expression of the Progesterone Receptor Membrane Component 1 (PGRMC1), a heme – binding protein with the ability to interact and stabilize epidermal growth factor receptor (EGFR) is frequently found in breast cancer tissue. Some evidence suggests that progesterone can stimulate and regulate breast cancer cell proliferation. The basis of the signaling mechanisms by which Progesterone exerts its function remains largely unknown. Both the nuclear and membrane progesterone receptors could play a significant role in the development and progression of breast cancer and both could become viable therapeutic options. We, aim to investigate the role of PGRMC1 in progesterone driven breast cancers. Materials and Methods: Human breast tissues were utilized to identify PGRMC1 expression along with a panel of normal and breast cancer cell lines. Two breast cancer cell lines (ZR-75-1 and MDA-MB-468) were selected and treated with progesterone and AG-205 (PGRMC1 inhibitor) at different concentrations to assess optimum dosage. We performed MTS assay, qRT-PCR, Western blot, immunofluorescence, immunohistochemistry and flow cytometry for measuring cell proliferation, apoptosis and key markers involved in these processes. We also performed an in silico analysis to compare the expression of PGRMC1 in various cell lines and breast cancer tissues. Results: Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal tissue. Increased PGRMC1 expression was observed specifically in ZR-75-1 and MDA-MB-468 cells by qRT-PCR, western blot and immunofluorescence, these results were validated and compared to microarray-based gene expression analysis of breast cell lines and breast tumor data sets. Progesterone treatment increased cell proliferation in a dose dependent manner while AG-205 decreased cell proliferation in a dose dependent manner in ZR-75-1 and MDA-MB-468. Minimal effects of AG-205 were observed in normal breast epithelial cells. AG-205 also, induced apoptosis in both ZR-75-1 and MDA-MB-468 cell lines. Furthermore, short-term treatment of progesterone increased both mRNA and protein expression of PGRMC1. Key markers of cell proliferation (pAKT, CCND1, pEGFR, pmTOR) and apoptosis (PTEN, Bcl2, Bax, Bim) revealed that PGRMC1 facilitated the proliferative effect of progesterone. Interestingly progesterone increases phosphorylation of EGFR and treatment of AG-205 alters EGFR expression in a dose dependent manner. Conclusion: Our data demonstrates that PGRMC1 plays a prominent role in regulating progesterone driven cell proliferation in both ER-positive and triple negative breast cancer cells. These initial findings uncover the potential of PGRMC1 as a therapeutic target for breast cancers. Citation Format: Pedroza DA, Subramani R, Galvez A, Lakshmanaswamy R. Progesterone drives ER-positive and triple negative breast cancer cell proliferation through progesterone receptor membrane component 1 [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-05-12.

MiR-199b-5p inhibits triple negative breast cancer cell proliferation, migration and invasion by targeting DDR1.

Triple negative breast cancer (TNBC) has received increasing attention from oncologists worldwide due to its poor prognosis and paucity of targeted therapies. MicroRNAs (miRs) are a group of small non-coding RNAs that are responsible for the post-transcriptional regulation of various target genes. The present study demonstrated that the expression of miR-199b-5p in breast cancer tissue was significantly reduced compared with that in normal breast tissues by reverse transcription-quantitative polymerase chain reaction. In addition, western blot analysis and luciferase reporter assays revealed that miR-199b-5p in TNBC cells inhibited discoidin domain receptor tyrosine kinase 1 expression by directly targeting its 3′-untranslated region. Furthermore, miR-199b-5p markedly suppressed the proliferation and invasion of TNBC cells, as demonstrated by using wound-healing, migration, invasion and proliferation assays. Collectively, these results indicate that miR-199b-5p may be a novel alternative therapeutic target for TNBC.

The SWI/SNF ATPases Are Required for Triple Negative Breast Cancer Cell Proliferation.

The Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi-subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells.

Abstract 3213: Rosehip (Rosa canina) extracts prevent MAPK and AKT-mediated cell proliferation in African American triple-negative breast cancer cells

Abstract Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer, characterized by its lack of the human epidermal growth factor receptor-2 (HER-2), the estrogen receptor (ER), and the progesterone receptor (PR). The high prevalence of triple negative tumors in young women is more commonly observed in young African American (AA) women. Currently, the existing targeted therapy is of minimal benefit in TNBCs. Akt and MAPK have been shown to promote cell proliferation in triple negative breast cancer and MAPK expression may be an underlying mechanism contributing to the generation of chemo-resistance in triple-negative breast cancer. Due to major concerns involving the occurrence of side effects and the emergence of drug-resistant cancer cells, there has been growing interest in the use of naturally occurring molecules with chemo-preventive and chemo- therapeutic properties in cancer treatment. Rosehip extracts have been used as dietary supplements to relieve symptoms associated with diarrhea, gastritis, and rheumatoid arthritis and have been shown to prevent cell proliferation in glioblastomas. This study investigated the efficacy of rosehip extracts in preventing cell proliferation in African American triple negative (HCC70, HCC1806) and luminal (HCC1500) breast cancer cell lines. Each of the breast cancer cell lines were treated with rosehip extracts (1mg/mL -25ng/mL) demonstrated a significant decrease in cell proliferation. The rosehip extract-mediated decrease in cell proliferation was equal to or better than the decrease of cell proliferation observed when known inhibitors of the MAPK (U0126, 10 μM) or AKT (LY294002, 20 μM) signaling pathways were utilized. Additionally, pretreatment of these cell lines with these Rosehip extracts (1mg/mL -25ng/mL) selectively decreased AKT, MAPK, p70S6K, and S6 phosphorylation suggesting these extracts prevent AA TNBC cell proliferation by blocking both the MAPK and AKT signaling mechanisms. Results from cell cycle analysis by flow cytometry, western blot analysis, as well as apoptosis studies demonstrate that rosehip extracts inhibit cell proliferation but do not promote apoptosis. Rosehip extracts also have a synergistic effect with Doxorubicin (20µM), a chemotherapeutic agent used to treat patients with breast cancer, in preventing triple negative breast cancer cell proliferation. Taken together these data suggest that rosehip extracts are capable of decreasing cell proliferation in African American triple negative breast cancer cells without promoting apoptosis and demonstrate a synergistic inhibition of cell proliferation with Doxorubicin, and rosehip extracts may serve as an alternative or compliment to current chemotherapeutic regimens for triple negative breast cancer. Citation Format: Patrice Cagle, Tonisha Coburn, Patrick M. Martin. Rosehip (Rosa canina) extracts prevent MAPK and AKT-mediated cell proliferation in African American triple-negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2014-3213

Abstract 3869: MicroRNA-200b blocks breast cancer lung metastasis in an orthotopic mouse mammary xenograft tumor model.

Abstract Triple negative, or basal-like, breast cancer is a highly invasive and metastatic form of breast cancer, and is difficult to treat due to the lack of target therapies. Epithelial to mesenchymal transition (EMT) refers to a developmental program that converts epithelial cells to mesenchymal cells, therefore enabling cells to migrate and invade. EMT is tightly controlled during development, and recent studies showed that EMT is often reactivated in cancer and plays an important role in cancer invasion and metastasis, and that EMT can also be regulated by microRNAs. MicroRNAs (miRs) are a large family of small non-coding RNAs that negatively regulate protein-coding gene expression posttranscriptionally. Although the miR-200 family members have been shown to be able to inhibit EMT, their effects on cancer metastasis continue to be controversial. We aim to investigate the effect of miR-200b on triple negative breast cancer lung metastasis and the underlying mechanism. It was found that stably expressing miR-200b in MDA-MB-231 cells blocked lung metastasis in an orthotopic mouse mammary xenograft tumor model. In vitro studies showed that stably expressing miR-200b significantly reduced triple negative breast cancer cell proliferation, migration, and invasion. Furthermore, stably expressing miR-200b also decreased tumor growth and angiogenesis in the orthotopic mouse mammary xenograft tumors. These findings suggest that miR-200b blocks breast cancer lung metastasis through its strong inhibitory effect on multiple-steps including proliferation, migration, invasion and angiogenesis. Citation Format: Brock Humphries, Zhishan Wang, Chengfeng Yang. MicroRNA-200b blocks breast cancer lung metastasis in an orthotopic mouse mammary xenograft tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3869. doi:10.1158/1538-7445.AM2013-3869

Abstract P6-14-04: Rosehip extracts prevent triple negative breast cancer cell proliferation by regulation the phosphorylation of p70S6 Kinase.

Abstract Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer, characterized by its lack of the human epidermal growth factor receptor-2 (HER-2), the estrogen receptor (ER), and the progesterone receptor (PR). Due to the lack of these hormone receptors, TNBC has abnormal therapeutic results; leading to the need for unique treatment methods. TNBC has a partial response to chemotherapy, which is associated with the lack of clinically established targeted therapies. Plant extracts have shown to be useful as chemopreventive agents due to their disruption of pro-survival and proliferative mechanisms on cancer cells. Rosehip extracts have shown potential anti-proliferative activity against cancer cells in vitro. This study investigated the effect of rosehip extracts in TNBC cell lines (HCC1806 and MB157) and basal-like breast cancer cell lines (HCC70 and HCC1500). To determine the inhibitory role of rosehip extracts in cell proliferation, MTT assays were conducted, following treatment with varying concentrations of rosehip extracts (1mg/ml, 250ug/ml, 25ug/ml, and 25ng/ml) for 48 hours. The data suggests that rosehip extracts, can significantly decrease cell proliferation in all the breast cancer cell lines assayed. Preliminary studies suggest that this is accomplished without toxic effects. Western blot analysis was used to investigate which signaling pathway rosehip extracts were affecting. Western Blot analysis suggests that rosehip extracts are inhibiting p70 S6 kinase phosphorylation and its ability to act as a kinase for S6 ribosomal protein. Based on these findings, rosehip extracts prevent cell proliferation by interfering with the p70 S6 kinase signaling. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-14-04.