Human Basal-like Breast Cancer Research Articles

MTA2 Enhances Breast Cancer Metastasis by Regulating the Rho Signaling Pathway.

Abstract Background: Metastasis is the ultimate cause of death for patients succumbing to breast cancer. The metastasis tumor associated (MTA) gene family has been linked with metastasis in breast cancer. Previously, we found that MTA2 overexpression enhanced the metastatic ability of the estrogen receptor-alpha (ERα)-negative cell line MDA-MB-231. This was accompanied by an increase in cell rounding, indicating a change in cytoskeletal organization. Here we explored the molecular pathways contributing to the MTA2-enhanced metastatic phenotype.Materials and Methods: MDA-MB-231 cells were engineered to overexpress MTA2. Changes in protein expression were determined by immunoblot assay. Differential gene expression was determined using Affymetrix microarrays and analyzed using dChip, BRB Array Tools, and R. Pathway analysis was carried out using the Database for Visualization, Annotation, and Integrated Discovery (DAVID). Validation was done by comparing to publicly available datasets. MTA2-overexpressing or control cells were transfected with a Rho GTP dissociation inhibitor alpha (Rho GDIα) or vector control expression constructs. Anchorage-independent growth was measured using soft-agar assays. Analysis of the impact of MTA2 and Rho GDIα combined expression was carried out using publically available datasets.Results: Analysis of enriched ontologies from significantly different genes between MTA2-overexpressing cells and clinical breast cancer samples of the basal subtype previously published by Richardson et al (1) indicated that the focal adhesion pathway was enhanced in MTA2-overexpressing samples. We found that a negative regulator of the focal adhesion pathway, Rho GDIα, was decreased in MTA2-overexpressing cells at the protein level. When a yellow fluorescent protein-tagged Rho GDIα expression vector was re-expressed in MTA2-overexpressing cells, there was a concomitant decrease in the expression of MTA2 and increased expression of endogenous Rho GDIα. When tested in soft agar assays, re-expression of Rho GDIα significantly reduced the colony-forming ability of MTA2-overexpressing cells. We examined the publically available dataset published by Wang et al. (2), and found that ERα-negative patients with high MTA2 expression and low Rho GDIa levels had earlier recurrence compared with all other patients (P=0.02).Conclusion: MTA2 overexpression was associated with an aggressive phenotype in MDA-MB-231 cells. These data indicate that MTA2 overexpression may be associated with increased signaling through the Rho pathway. We found that MTA2 and Rho GDIα form a regulatory loop which reinforces either MTA2 high or MTA2 low expression levels. Furthermore, we found that combined high MTA2 and low Rho GDIα expression had a negative impact on patient outcomes in the ERα-negative population. We are currently exploring the use of Rho pathway inhibitors for their potential to block this phenotype, and to explore the MTA2/Rho GDIα regulatory loop.1) Richardson AL, Wang ZC, De Nicolo A, Lu X et al. X chromosomal abnormalities in basal-like human breast cancer. Cancer Cell 2006 Feb;9(2):121-32.2) Wang Y, Klijn JG, Zhang Y, Sieuwerts AM et al. Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 2005 Feb 19-25;365(9460):671-9. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6143.

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 (>55% inhibition, P<0.01 vs. control). For GILM2 tumors, 25 and 75 mg/kg motesanib gave 47% and 58% growth inhibition, respectively (P<0.008), and for HCC1187, these doses produced 81% and 91% growth inhibition, respectively (P<0.001). GILM2 tumors from mice receiving >25mg/kg had smaller viable tissue areas (P<0.001), and fewer CD-31 positive blood vessels (P<0.001) than tumors from control mice. Immunostaining showed that 25mg/kg motesanib reduced proliferation of GILM2 tumors (P<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<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<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.

Transcriptional activation of the Lats1 tumor suppressor gene in tumors of CUX1 transgenic mice

BackgroundLats1 (large tumor suppressor 1) codes for a serine/threonine kinase that plays a role in the progression through mitosis. Genetic studies demonstrated that the loss of LATS1 in mouse, and of its ortholog wts (warts) in Drosophila, is associated with increased cancer incidence. There are conflicting reports, however, as to whether overexpression of Lats1 inhibits cell proliferation. CUX1 is a transcription factor that exists in different isoforms as a result of proteolytic processing or alternative transcription initiation. Expression of p110 and p75 CUX1 in transgenic mice increases the susceptibility to cancer in various organs and tissues. In tissue culture, p110 CUX1 was shown to accelerate entry into S phase and stimulate cell proliferation.ResultsGenome-wide location arrays in cell lines of various cell types revealed that Lats1 was a transcriptional target of CUX1. Scanning ChIP analysis confirmed that CUX1 binds to the immediate promoter of Lats1. Expression of Lats1 was reduced in cux1-/- MEFs, whereas it was increased in cells stably or transiently expressing p110 or p75 CUX1. Reporter assays confirmed that the immediate promoter of Lats1 was sufficient to confer transcriptional activation by CUX1. Lats1 was found to be overexpressed in tumors from the mammary gland, uterus and spleen that arise in p110 or p75 CUX1 transgenic mice. In tissue culture, such elevated LATS1 expression did not hinder cell cycle progression in cells overexpressing p110 CUX1.ConclusionWhile inactivation of Lats1/wts in mouse and Drosophila can increase cancer incidence, results from the present study demonstrate that Lats1 is a transcriptional target of CUX1 that can be overexpressed in tumors of various tissue-types. Interestingly, two other studies documented the overexpression of LATS1 in human cervical cancers and basal-like breast cancers. We conclude that, similarly to other genes involved in mitotic checkpoint, cancer can be associated with either loss-of-function or overexpression of Lats1.

A Novel Lung Metastasis Signature Links Wnt Signaling with Cancer Cell Self-Renewal and Epithelial-Mesenchymal Transition in Basal-like Breast Cancer

The establishment of metastasis depends on the ability of cancer cells to acquire a migratory phenotype combined with their capacity to recreate a secondary tumor in a distant tissue. In epithelial cancers, such as those of the breast, the epithelial-mesenchymal transition (EMT) is associated with basal-like breast cancers, generates cells with stem-like properties, and enables cancer cell dissemination and metastasis. However, the molecular mechanism(s) that connects stem cell-like characteristics with EMT has yet to be defined. Using an orthotopic model of human breast cancer metastasis to lung, we identified a poor prognosis gene signature, in which several components of the wnt signaling pathway were overexpressed in early lung metastases. The wnt genes identified in this signature were strongly associated with human basal-like breast cancers. We found that inhibiting wnt signaling through LRP6 reduced the capacity of cancer cells to self-renew and seed tumors in vivo. Furthermore, inhibition of wnt signaling resulted in the reexpression of breast epithelial differentiation markers and repression of EMT transcription factors SLUG and TWIST. Collectively, these results provide a molecular link between self-renewal, EMT, and metastasis in basal-like breast cancers.

Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression

TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERalpha and ERbeta) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17beta-hydroxysteroid dehydrogenases (17betaHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERalpha-positive luminal MCF7 breast cancer cells. Low levels of ERalpha and ERbeta mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17betaHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17betaHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17betaHSD1, and 17betaHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERalpha, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERbeta, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.

Igf1r as a therapeutic target in a mouse model of basal-like breast cancer

Considering the strong association between dysregulated insulin-like growth factor (IGF) signaling and various human cancers, we have used an expedient combination of genetic analysis and pharmacological treatment to evaluate the potential of the type 1 IGF receptor (Igf1r) for targeted anticancer therapy in a mouse model of mammary tumorigenesis. In this particular strain of genetically modified animals, histopathologically heterogeneous invasive carcinomas exhibiting up-regulation of the Igf1r gene developed extremely rapidly by mammary gland-specific overexpression of constitutively active oncogenic Kras* (mutant Kras(G12D)). Immunophenotyping data and expression profiling analyses showed that, except for a minor luminal component, these mouse tumors resembled basal-like human breast cancers. This is a group of aggressive tumors of poor prognosis for which there is no targeted therapy currently available, and it includes a subtype correlating with KRAS locus amplification. Conditional ablation of Igf1r in the mouse mammary epithelium increased the latency of Kras*-induced tumors very significantly (approximately 11-fold in comparison with the intact model), whereas treatment of tumor-bearing animals by administration of picropodophyllin (PPP), a specific Igf1r inhibitor, resulted in a dramatic decrease in tumor mass of the main forms of basal-like carcinomas. PPP also was effective against xenografts of the human basal-like cancer cell line MDA-MB-231, which carries a KRAS(G13D) mutation.

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.
 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.
 Results: According to MTT assay, AMI could obviously inhibit the proliferation of the MDA-MB-468 cells (p<0.05) and the effect was stronger when combined with Tarceva (p<0.01); When the PTEN gene was knocked down, AMI only combined with Tarceva could inhibit the cells' proliferation (p<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<0.001) and down-regulate the p53 (p<0.01), MDM2 (p<0.01), Akt2 (p<0.001) and PIP (p<0.001) gene expressions. In-cell western assay indicated that p53 protein was down-regulated at 15min(p<0.05) and 1h(p<0.001), and p-Akt protein expression showed significant down-regulation at 15min(p<0.01), 30min(p<0.001) and 2d(p<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<0.05, p<0.005). After PTEN SiRNA intervene, AMI down-regulate p53 (p<0.001), p-Akt (p<0.005) and EGFR (p<0.05) protein expressions; and its down-regulation effect to p-Akt reduced, but increased to p53, and EGFR.
 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.

Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1 -mutated basal-like breast cancer

Women carrying germ-line mutations in BRCA1 are strongly predisposed to developing breast cancers with characteristic features also observed in sporadic basal-like breast cancers. They appear as high-grade tumors with high proliferation rates and pushing borders. On the molecular level, they are negative for hormone receptors and ERBB2, display frequent TP53 mutations, and express basal epithelial markers. To study the role of BRCA1 and P53 loss of function in breast cancer development, we generated conditional mouse models with tissue-specific mutation of Brca1 and/or p53 in basal epithelial cells. Somatic loss of both BRCA1 and p53 resulted in the rapid and efficient formation of highly proliferative, poorly differentiated, estrogen receptor-negative mammary carcinomas with pushing borders and increased expression of basal epithelial markers, reminiscent of human basal-like breast cancer. BRCA1- and p53-deficient mouse mammary tumors exhibit dramatic genomic instability, and their molecular signatures resemble those of human BRCA1-mutated breast cancers. Thus, these tumors display important hallmarks of hereditary breast cancers in BRCA1-mutation carriers.

Mesenchyme Forkhead 1 ( FOXC2 ) plays a key role in metastasis and is associated with aggressive basal-like breast cancers

The metastatic spread of epithelial cancer cells from the primary tumor to distant organs mimics the cell migrations that occur during embryogenesis. Using gene expression profiling, we have found that the FOXC2 transcription factor, which is involved in specifying mesenchymal cell fate during embryogenesis, is associated with the metastatic capabilities of cancer cells. FOXC2 expression is required for the ability of murine mammary carcinoma cells to metastasize to the lung, and overexpression of FOXC2 enhances the metastatic ability of mouse mammary carcinoma cells. We show that FOXC2 expression is induced in cells undergoing epithelial-mesenchymal transitions (EMTs) triggered by a number of signals, including TGF-beta1 and several EMT-inducing transcription factors, such as Snail, Twist, and Goosecoid. FOXC2 specifically promotes mesenchymal differentiation during an EMT and may serve as a key mediator to orchestrate the mesenchymal component of the EMT program. Expression of FOXC2 is significantly correlated with the highly aggressive basal-like subtype of human breast cancers. These observations indicate that FOXC2 plays a central role in promoting invasion and metastasis and that it may prove to be a highly specific molecular marker for human basal-like breast cancers.

Serial transplantation of NMU‐induced rat mammary tumors: A model of human breast cancer progression

Human breast cancer is a heterogeneous disease that appears to progress from an in situ tumor to invasive cancer. Little is known about the molecular events driving this progression. Although microarray technology has helped us understand the genetic heterogeneity of breast cancer, its application to studying the transition from in situ to invasive disease is limited by the inability to follow the progression of a single patient's tumor. We previously used rat specific microarrays to show that N-methyl-N-nitrosourea induced tumors are similar to low-grade estrogen-receptor positive human breast cancer. Here, we transplanted these tumors through 5 generations of syngeneic hosts, and studied 65 resulting tumors. Most transplanted tumors gradually progressed from a noninvasive, low-grade cancer to a higher-grade invasive disease, losing p63 localization and basement membrane integrity. Invasive cancers frequently demonstrated a more mesenchymal phenotype with increased vimentin expression. Additionally, a unique transplant series is described with a phenotype similar to human basal-like breast cancer. Rat-specific Affymetrix gene arrays containing 15,866 gene probes identified genes that differentiated highly invasive tumors from those of low invasive potential. A linear regression analysis was used to find genes whose change in expression paralleled increasing invasive features independent of the transplant lineage of origin. Genes identified were assigned membership in cell adhesion, signal transduction, cell cycle and extracellular matrix groups, among others. This animal model overcomes the difficulty in studying human breast cancer progression. Our data support a gradual and continuous alteration in programs of gene expression during breast cancer invasion.

X chromosomal abnormalities in basal-like human breast cancer

Sporadic basal-like cancers (BLC) are a distinct class of human breast cancers that are phenotypically similar to BRCA1-associated cancers. Like BRCA1-deficient tumors, most BLC lack markers of a normal inactive X chromosome (Xi). Duplication of the active X chromosome and loss of Xi characterized almost half of BLC cases tested. Others contained biparental but nonheterochromatinized X chromosomes or gains of X chromosomal DNA. These abnormalities did not lead to a global increase in X chromosome transcription but were associated with overexpression of a small subset of X chromosomal genes. Other, equally aneuploid, but non-BLC rarely displayed these X chromosome abnormalities. These results suggest that X chromosome abnormalities contribute to the pathogenesis of BLC, both inherited and sporadic.