Human Basal-like Breast Cancer Cell Research Articles

FAK activates AKT-mTOR signaling to promote the growth and progression of MMTV-Wnt1-driven basal-like mammary tumors

BackgroundBreast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer.MethodsTo understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested.ResultsWe found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival.ConclusionsIn summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target.

Glyoxalase 1 gene is highly expressed in basal-like human breast cancers and contributes to survival of ALDH1-positive breast cancer stem cells.

Glyoxalase 1 (GLO1) is a ubiquitous enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis that induces apoptosis. In this study, we found that GLO1 gene expression correlates with neoplasm histologic grade (χ2 test, p = 0.002) and is elevated in human basal-like breast cancer tissues. Approximately 90% of basal-like cancers were grade 3 tumors highly expressing both GLO1 and the cancer stem cell marker ALDH1A3. ALDH1high cells derived from the MDA-MB 157 and MDA-MB 468 human basal-like breast cancer cell lines showed elevated GLO1 activity. GLO1 inhibition using TLSC702 suppressed ALDH1high cell viability as well as the formation of tumor-spheres by ALDH1high cells. GLO1 knockdown using specific siRNAs also suppressed ALDH1high cell viability, and both TLSC702 and GLO1 siRNA induced apoptosis in ALDH1high cells. These results suggest GLO1 is essential for the survival of ALDH1-positive breast cancer stem cells. We therefore conclude that GLO1 is a potential therapeutic target for treatment of basal-like breast cancers.

Abstract 1923: Claudin 1 expression levels affect miRNA dynamics in human basal-like breast cancer cells

Abstract MiRNAs have been shown to regulate numerous cellular functions including metastasis of cancer cells, and are considered promising biomarkers of disease. The major tight junction protein of epithelial cells, claudin 1, is down regulated or absent in human invasive breast cancer and is therefore considered a putative tumor suppressor. However, claudin 1 has now been shown to be highly expressed in the basal-like molecular subtype and in vitro studies revealed that claudin 1 can regulate breast cancer cell motility and proliferation. To gain further understanding of how claudin 1 mediates its activities in breast cancer, we examined changes in global micro RNA (miRNA) gene expression when claudin 1 was over-expressed in the phenotypically basal-like human breast cancer (HBC) cell line MDA-MB231. Using next generation sequencing, followed by qPCR validation, we identified seven miRNAs (miR-9-5p, miR-9-3p, miR- let-7c, miR-127-3p, miR-99a-5p, miR-129-5p, and miR-146a-5p) whose expression were altered as a consequence of claudin 1 over expression. Most of these miRNAs were down regulated and associated with tumor suppression in a variety of cancers including breast. Additionally, using gene expression profiling analysis of the claudin 1 over expressing clones, we identified a number of down regulated EMT related genes, including PDGFRB and CDH1 (E- cadherin). Expression of these genes is frequently lost during breast tumor progression. Interestingly, SPP1 and SERPINE 1, genes often associated with tumor cell migration and motility, were up regulated in the claudin 1 over expressing clones. Employing the miRNA target prediction program miRWalk, a number of validated miRNA target sites were identified on some of these deregulated genes. A target site for miR-9-5p was identified on the CDH1 sequence, whereas a target site of miR-99-5p was identified on SERPINE 1. Altogether, over 500 target genes have been validated as targets for the deregulated miRNAs. Overall, we show for the first time that in HBC, claudin 1 can alter the dynamics of a number of miRNAs involved in tumor progression. Moreover, our data provides further evidence to suggest that claudin 1 has the potential to be a useful biomarker that identifies a subset of tumors, within the aggressive but currently poorly characterized basal-like subtype. Our study also suggests that miRNAs may be used in conjunction with claudin 1 to better characterize these cancers. Further studies are now warranted to determine the role of these miRNAs in facilitating the function of claudin 1 in breast cancer. Citation Format: Anne AA Blanchard, Anna Majer, Sarah Medina, Stephanie A. Booth, Yvonne Myal. Claudin 1 expression levels affect miRNA dynamics in human basal-like breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1923.

Anti-apoptotic mechanism for p21(waf1) in human basal like breast cancer cell line HCC1937

To explore the anti-apoptotic mechanism of p21(waf1) in human basal like breast cancer cell line HCC1937. There were 3 groups, i.e. experimental group HCC1937 with lentivirus -p21(waf1)-shRNA-RFP, control group 1 HCC1937 without lentivirus and control group 2 HCC1937 with lentivirus -RFP. The p21(waf1) and bim mRNA and protein expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. And apoptosis of HCC1937 in different groups was assayed by terminal deoxynucleotidyl transferase mediated C-dUTP nick end labeling (TUNEL). After interference with lentivirus-p21(waf1)-shRNA-RFP, p21(waf1) mRNA and protein expressions declined significantly in the experimental group versus the control groups (experiment group: 0.260 ± 0.004, 0.293 ± 0.006, control group 1: 0.879 ± 0.028, 0.483 ± 0.071, control group 2: 0.870 ± 0.025, 0.469 ± 0.047, all P < 0.01). The bim mRNA and protein expressions increased. And there was significant difference between the experiment and control groups (experiment group: 0.420 ± 0.013, 0.355 ± 0.007, control group 1: 0.258 ± 0.005, 0.142 ± 0.012, control group 2: 0.259 ± 0.002, 0.147 ± 0.013, all P < 0.001); apoptotic index increased (experiment group: 0.279 ± 0.012, control group 1: 0.145 ± 0.008, control group 2: 0.148 ± 0.012, both P < 0.001). In human basal-like breast cancer cell line HCC1937, p21(waf1) exerts anti-apoptotic activity by inhibiting the expression of bim, a mediator of mitochondrial apoptosis.

Ets-1 is a transcriptional mediator of oncogenic nitric oxide signaling in estrogen receptor-negative breast cancer

IntroductionThe Ets-1 transcription factor is a candidate breast cancer oncogene that regulates the expression of genes involved in tumor progression and metastasis. Ets-1 signaling has also been linked to the development of a basal-like breast cancer phenotype. We recently described a nitric oxide (NO)-induced gene signature that is associated with poor disease outcome in estrogen receptor-negative (ER-) breast cancer and contains both stem cell-like and basal-like components. Thus, we examined the role of Ets-1 in NO signaling and NO-induced phenotypes in ER- human breast cancer cells.MethodsPromoter region analyses were performed on genes upregulated in inducible nitric oxide synthase (NOS2) high expressing tumors for Ets-binding sites. In vitro mechanisms were examined in human basal-like breast cancer cells lines. NO signaling effects were studied using either forced NOS2 expression or the use of a chemical NO-donor, diethlylenetriamine NONOate (DETANO).ResultsPromoter region analysis of genes that are up-regulated in human ER-negative breast tumors with high NOS2 expression revealed that the Ets-binding sequence is the only common promoter element present in all of these genes, indicating that Ets-1 is the key transcriptional factor down-stream of oncogenic NOS2-signaling. Accordingly, both forced NOS2 over-expression and exposure to NO-donors resulted in significant Ets-1 transcriptional activation in ER- breast cancer cells. Functional studies showed that NO activated Ets-1 transcriptional activity via a Ras/MEK/ERK signaling pathway by a mechanism that involved Ras S-nitrosylation. RNA knock-down of Ets-1 suppressed NO-induced expression of selected basal-like breast cancer markers such as P-cadherin, S100A8, IL-8 and αβ-crystallin. Additionally, Ets-1 knock-down reduced NO-mediated cellular proliferation, matrix metalloproteinase and cathepsin B activities, as well as matrigel invasion.ConclusionsThese data show that Ets-1 is a key transcriptional mediator of oncogenic NO signaling that promotes the development of an aggressive disease phenotype in ER- breast cancer in an Ets-1 and Ras-dependent manner, providing novel clues of how NOS2 expression in human breast tumors is functionally linked to poor patient survival.

FOXC1 regulates the functions of human basal-like breast cancer cells by activating NF-κB signaling

Human basal-like breast cancer (BLBC) is an enigmatic and aggressive malignancy with a poor prognosis. There is an urgent need to identify therapeutic targets for BLBC, because current treatment modalities are limited and not effective. The forkhead box transcription factor FOXC1 has recently been identified as a critical functional biomarker for BLBC. However, how it orchestrates BLBC cells was not clear. Here we show that FOXC1 activates the transcription factor nuclear factor-κB (NF-κB) in BLBC cells by increasing p65/RelA protein stability. High NF-κB activity has been associated with estrogen receptor-negative breast cancer, particularly BLBC. The effect of FOXC1 on p65/RelA protein stability is mediated by increased expression of Pin1, a peptidyl-prolyl isomerase. FOXC1 requires NF-κB for its regulation of cell proliferation, migration and invasion. Notably, FOXC1 overexpression renders breast cancer cells more susceptible to pharmacological inhibition of NF-κB. These results suggest that BLBC cells may rely on FOXC1-driven NF-κB signaling. Interventions of this pathway may provide modalities for the treatment of BLBC.

The Anti-Tumor Effect of Motesanib in Pre-Clinical Models of Human Basal-Like Breast Cancer.

Abstract Background: Growth of breast cancer is dependent upon sustained angiogenesis, of which vascular endothelial growth factor (VEGF) is a major stimulant. Increased expression of VEGF in breast cancers is associated with poor prognosis and disease recurrence, and anti-angiogenic agents are being developed as potential new therapies. Motesanib is an oral inhibitor of VEGF receptors 1, 2 and 3, platelet-derived growth factor receptor, and stem cell factor receptor (c-Kit). This study determined the effects of motesanib on the growth of human basal-like breast cancer cell lines in immunodeficient mice, alone and in combination with paclitaxel.Materials and Methods: Breast cancer cells BT-549 and GILM2 were injected into the mammary fatpad (MFP) of mice; HCC1187 cells were injected on the subcutaneous flank. Mice with established tumors (150-200 mm3) received motesanib orally BID, at 7.5, 25 or 75 mg/kg for three weeks. Tumors were collected for analysis of blood vessels (CD-31 staining), viable tumor area, and for markers of proliferation (Ki67) and apoptosis (cleaved caspase-3). GILM2 cells were injected into the tibia of mice to model growth in bone; mice with GILM2 MFP or tibia tumors received motesanib (25 mg/kg BID) or paclitaxel (24 mg/kg Q4Dx5), or the combination. Intratibial GILM2 tumors expressing luciferase were monitored by bioluminescence and radiography to evaluate osteolysis. Analyses of tumor size were assessed by ANOVA, and immunohistochemical tests by Student's t tests.Results: Motesanib produced dose-dependent inhibition of growth of BT-549, GILM2 and HCC1187 tumors. All doses of motesanib significantly inhibited the growth of BT-549 tumors (&amp;gt;55% inhibition, P&amp;lt;0.01 vs. control). For GILM2 tumors, 25 and 75 mg/kg motesanib gave 47% and 58% growth inhibition, respectively (P&amp;lt;0.008), and for HCC1187, these doses produced 81% and 91% growth inhibition, respectively (P&amp;lt;0.001). GILM2 tumors from mice receiving &amp;gt;25mg/kg had smaller viable tissue areas (P&amp;lt;0.001), and fewer CD-31 positive blood vessels (P&amp;lt;0.001) than tumors from control mice. Immunostaining showed that 25mg/kg motesanib reduced proliferation of GILM2 tumors (P&amp;lt;0.001 vs. control), with no change in numbers of apoptotic cells. Treatment with 25 mg/kg motesanib or paclitaxel (24mg/kg Q4Dx5) significantly reduced GILM2 tumor burden in the MFP (46% and 49% inhibition, respectively) and in the bone (77% and 68% inhibition, respectively) (P&amp;lt;0.05 vs. control). Combining the agents did not enhance the anti-tumor effects of paclitaxel. Bioluminescent measurements and X-rays showed that motesanib alone was as effective as paclitaxel at reducing GILM2 growth and osteolytic destruction in bone (P&amp;lt;0.05, vs. control).Conclusion: Oral administration of motesanib significantly inhibited the growth of three ER-, PR- HER2-negative basal-like breast cancer cell lines in vivo. Motesanib was as effective as paclitaxel at controlling the growth of GILM2 in the MFP, and reduced osteolysis in bone tumors. These data suggest that motesanib has anti-angiogenic action in diverse tumor microenvironments. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5057.

The mechanism of Astragalus membranaceus' effects on the proliferation of human basal-like breast cancer cell line MDA-MB-468 from p53/MDM2 pathway.

Abstract Abstract #2116 Background: Based on the expression of ER,PR and Her2, breast cancer is classified into several subtypes. Thereinto, basal-like type ("triple-negative" phenotype) is associated with poor prognosis. Patients with this type are unlikely to benefit from currently available targeted therapy. And some researches revealed traditional Chinese medicine could play an important role in breast cancer treatment. p53 mutations can be found in most of basal-like breast cancer patients and this type's cell lines. In our study, we mainly focused on the mechanism of Astragalus membranaceus' effect on the proliferation of MDA-MB-468 cell from p53/MDM2 pathway.&amp;#x2028; Material and Methods: The MDA-MB-468 cells were intervened by Astragalus membranaceus Injection (AMI) which contains 1g/ml crude drugs. The effect of AMI on the proliferation of the MDA-MB-468 cells was detected by MTT assay and its time-effect relationship was observed by cell counting kit-8 (CCK-8) assay. At two days after AMI intervention, the mRNA expression of some indicators were assessed by Real-time Quantitative PCR, including p53, MDM2, EGFR, PIP, PI3K, Akt1, Akt2 and PTEN. The anti-proliferation effect of AMI on the cells proliferation was detected by MTT assay after PTEN gene was knocked down by the small interfering RNA (SiRNA), and the protein expressions of EGFR, p-Akt, MDM2 and p53 were obtained by in-cell western assay.&amp;#x2028; Results: According to MTT assay, AMI could obviously inhibit the proliferation of the MDA-MB-468 cells (p&amp;lt;0.05) and the effect was stronger when combined with Tarceva (p&amp;lt;0.01); When the PTEN gene was knocked down, AMI only combined with Tarceva could inhibit the cells' proliferation (p&amp;lt;0.005). CCK-8 assay showed that the anti-proliferation effect of AMI was positively correlated with the intervention duration. Real-time Quantitative PCR revealed that AMI could up-regulate the PTEN gene expression (p&amp;lt;0.001) and down-regulate the p53 (p&amp;lt;0.01), MDM2 (p&amp;lt;0.01), Akt2 (p&amp;lt;0.001) and PIP (p&amp;lt;0.001) gene expressions. In-cell western assay indicated that p53 protein was down-regulated at 15min(p&amp;lt;0.05) and 1h(p&amp;lt;0.001), and p-Akt protein expression showed significant down-regulation at 15min(p&amp;lt;0.01), 30min(p&amp;lt;0.001) and 2d(p&amp;lt;0.001) after AMI intervene; when AMI combined with Tarceva, the duration of the down-regulation effect to p53 and p-Akt were all prolonged, and EGFR and MDM2 expressions were down-regulated(p&amp;lt;0.05, p&amp;lt;0.005). After PTEN SiRNA intervene, AMI down-regulate p53 (p&amp;lt;0.001), p-Akt (p&amp;lt;0.005) and EGFR (p&amp;lt;0.05) protein expressions; and its down-regulation effect to p-Akt reduced, but increased to p53, and EGFR.&amp;#x2028; Discussion: AMI can significantly inhibit the proliferation of MDA-MB-468 cells and present a time-dependent manner. The effect became stronger after combined with Tarceva. The main mechanism of its anti-proliferation effect may be to activate the positive feedback loop of the p53/ MDM2 pathway. It negatively regulates the PI3K by up-regulating the PTEN gene expression to promote the phosphorylation at D3 point of PIP3, so Akt's phosphorylation level is down-regulated and the cells proliferation was inhibited. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2116.

Mammary Tumors Initiated by Constitutive Cdk2 Activation Contain an Invasive Basal-like Component

The basal-like subtype of breast cancer is associated with invasiveness, high rates of postsurgical recurrence, and poor prognosis. Aside from inactivation of the BRCA1 tumor-suppressor gene, little is known concerning the mechanisms that cause basal breast cancer or the mechanisms responsible for its invasiveness. Here, we show that the heterogeneous mouse mammary tumor virus-cyclin D1-Cdk2 (MMTV-D1K2) transgenic mouse mammary tumors contain regions of spindle-shaped cells expressing both luminal and myoepithelial markers. Cell lines cultured from these tumors exhibit the same luminal/myoepithelial mixed-lineage phenotype that is associated with human basal-like breast cancer and express a number of myoepithelial markers including cytokeratin 14, P-cadherin, α smooth muscle actin, and nestin. The MMTV-D1K2 tumor-derived cell lines form highly invasive tumors when injected into mouse mammary glands. Invasion is associated with E-cadherin localization to the cytoplasm or loss of E-cadherin expression. Cytoplasmic E-cadherin correlates with lack of colony formation in vitro and β-catenin and p120ctn localization to the cytoplasm. The data suggest that the invasiveness of these cell lines results from a combination of factors including mislocalization of E-cadherin, β-catenin, and p120ctn to the cytoplasm. Nestin expression and E-cadherin mislocalization were also observed in human basal-like breast cancer cell lines, suggesting that these results are relevant to human tumors. Together, these results suggest that abnormal Cdk2 activation may contribute to the formation of basal-like breast cancers.