Luminal Human Breast Cancer Cell Research Articles

Down-regulation of the Tumor Suppressor CYLD Enhances the Transformed Phenotype of Human Breast Cancer Cells.

The cylindromatosis tumor suppressor (CYLD) has been implicated in the inhibition of human breast cancer development by virtue of the poor prognosis of patients with down-regulated CYLD expression. In order to investigate the mechanism of breast cancer suppression by CYLD, in the present study, cellular and molecular aspects of CYLD-dependent phenotypic regulation of different types of human breast cancer cell lines were analyzed. CYLD expression was down-regulated by RNA interference in human breast cancer cell lines. Parental and CYLD-deficient cell lines were evaluated for their viability, migratory capacity, anchorage-independent growth and chemoresistance. Wild-type and mutated forms of CYLD were also evaluated for their ability to suppress the clonogenic potential of breast cancer cells. CYLD down-regulation enhanced the survival and migratory properties of basal and luminal breast cancer cell lines. In addition, down-regulation of CYLD expression enhanced the ability of human breast cancer cells to grow in an anchorage-independent manner and could be associated with resistance to chemotherapeutic drugs. The growth-suppressive properties of CYLD on breast cancer cell lines were dependent on its de-ubiquitinating activity and its amino terminal cytoskeleton-interacting region. Our results establish a broad range of tumor-suppressive properties that are conferred by CYLD in basal and luminal human breast cancer cells and support the significance of targeted de-ubiquitination by CYLD in breast cancer cell growth suppression.

Anticancer effects of tributyltin chloride and triphenyltin chloride in human breast cancer cell lines MCF-7 and MDA-MB-231.

Triorganotin compounds induce hormonal alterations, i.e., endocrine-disrupting effects in mammals, including humans. Tributyltin chloride (TBT-Cl) and triphenyltin chloride (TPT-Cl) are known to function as nuclear retinoid X receptor (RXR) agonists. Their cytotoxic effects in ER(+) luminal human breast cancer cell line MCF-7 and ER(-) basal-like human breast cancer cell line MDA-MB-231 were examined. We observed significantly higher toxicity of TBT-Cl in comparison with TPT-Cl in both cell lines. Comparable apoptosis-inducing concentrations were 200 and 800nM, respectively, as shown by PARP cleavage and FDA staining. Both compounds activated executive caspases in the concentration-dependent manner in MDA-MB-231 cells, but the onset of TPT-Cl-induced caspase-3/7 activation was delayed in comparison with TBT-Cl. Both compounds slowed down the migration of these highly invasive cells, which was accompanied by RARbeta upregulation. Other RAR and RXR expressions were differentially modulated by studied organotins in both cell lines.

Abstract P2-04-05: Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones

Abstract All Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+PR+ ones. We have identified two such Luminal-derived ER–PR– cells: The first, we call "Luminobasal" (LB), are ER–PR– and cytokeratin 5 (CK5)-positive. The second, we call "Double Negative" (DN), are ER–PR– and CK5–. Currently, neither is targeted for treatment. Luminobasal cells: To address the relationships between true Luminal ER+PR+CK5– and Luminobasal ER–PR–CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from solid tumor xenografts starting from the same parental T47Dco Luminal human breast cancer cell line. Cells were gene profiled and unique immunohistochemical (IHC) biomarkers for each were confirmed. Interaction dynamics studies show that in mixed-cell 3D colonies, pLUM or Luminal MCF-7 cells suppress growth of pLB cells. Similarly, in mixed-cell solid tumor xenografts, pLUM cells suppress pLB proliferation. Alarmingly, in mixed-cell models, monotherapy of a pLUM or MCF-7 subpopulation with the antiestrogen Fulvestrant inadvertently expands the number of pLB cells. This suggested that pLB cells also need to be treated. An 89 drug FDA-approved oncology library and high throughput screening methods were therefore used to show that pLB cells are specifically targeted by the EGFR inhibitors Gefitinib and Erlotinib. We then showed, in both mixed-cell 3D colonies and mixed-cell solid mouse tumors, that combination therapy using Fulvestrant plus Gefitinib constitutes a robust treatment strategy that targets both cell populations simultaneously. Double Negative cells: DN cells are EGFR– and therefore unaffected by EGFRi. However they express the Luminal marker Claudin-3 (CLD3). Among other conditions, DN cells arise as a discrete CLD3+ subpopulation within CLD3– pLB xenografts. This allowed us to use CLD3 for FACS isolation and purification of DN cells from xenografts. Purified cells were gene expression profiled, which showed that DN cells have a unique genomic signature that distinguishes them from LUM and LB cells. They express diagnostic markers suitable for IHC and FACS-sorting, some of which also present potential therapeutic targets. We propose that heterogeneous Luminal breast cancers may be best treated with combination therapies that include endocrine, EGFRi and/or DN inhibitors. Importantly, optimizing combination regimens that eradicate or suppress not only ER+PR+ cells but also diverse ER–PR– cells in Luminal disease requires that primary tumors be pre-screened for appropriate biomarkers, including but not limited to ER, PR, CK5 and EGFR. Citation Format: Allison L Scaling, Aaron J Knox, Maurcio P Pinto, Brian S Bliesner, James M Haughian, Hany A Abdel-Hafiz, Kathryn B Horwitz. Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-05.

Modeling luminal breast cancer heterogeneity: combination therapy to suppress a hormone receptor-negative, cytokeratin 5-positive subpopulation in luminal disease.

IntroductionMany Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+ PR+ ones. One such subpopulation we call “Luminobasal” is ER-, PR- and cytokeratin 5 (CK5)-positive. It is not targeted for treatment.MethodsTo address the relationships between ER+PR+CK5– and ER–PR–CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from the same parental Luminal human breast cancer cell line. We used high-throughput screening to identify pLB-specific drugs and examined their efficacy alone and in combination with hormone therapy in mixed-cell tumor models.ResultsWe show that pLUM and MCF7 cells suppress proliferation of pLB cells in mixed-cell 3D colonies in vitro and that pLUM cells suppress growth of pLB cells in mixed-cell xenografts in vivo. High-throughput screening of 89 FDA-approved oncology drugs shows that pLB cells are sensitive to monotherapy with the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib. By exploiting mixed-cell 3D colonies and mixed-cell solid mouse tumors models we demonstrate that combination therapy with gefitinib plus the anti-estrogen fulvestrant constitutes a robust treatment strategy.ConclusionsWe propose that response to combination endocrine/EGFR inhibitor therapies in heterogeneous Luminal cancers may improve long-term survival in patients whose primary tumors have been preselected for appropriate biomarkers, including ER, PR, CK5 and EGFR.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0418-6) contains supplementary material, which is available to authorized users.

SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation

Activated Cdc42-associated kinase 1 (ACK1) is a nonreceptor tyrosine kinase linked to cellular transformation. The aberrant regulation of ACK1 promotes tumor progression and metastasis. Therefore, ACK1 is regarded as a valid target in cancer therapy. Seven in absentia homolog (SIAH) ubiquitin ligases facilitate substrate ubiquitinylation that targets proteins to the proteasomal degradation pathway. Here we report that ACK1 and SIAH1 from Homo sapiens interact in a yeast two-hybrid screen. Protein-protein interaction studies and protein degradation analyses using deletion and point mutants of ACK1 verify that SIAH1 and the related SIAH2 interact with ACK1. The association between SIAHs and ACK1 depends on the integrity of a highly conserved SIAH-binding motif located in the far C-terminus of ACK1. Furthermore, we demonstrate that the interaction of ACK1 with SIAH1 and the induction of proteasomal degradation of ACK1 by SIAH1 are independent of ACK1's kinase activity. Chemical inhibitors blocking proteasomal activity corroborate that SIAH1 and SIAH2 destabilize the ACK1 protein by inducing its proteasomal turnover. This mechanism apparently differs from the lysosomal pathway targeting ACK1 after stimulation with the epidermal growth factor. Our data also show that ACK1, but not ACK1 mutants lacking the SIAH binding motif, has a discernable negative effect on SIAH levels. Additionally, knockdown approaches targeting the SIAH2 mRNA uncover specifically that the induction of SIAH2 expression, by hormonally-induced estrogen receptor (ER) activation, decreases the levels of ACK1 in luminal human breast cancer cells. Collectively, our data provide novel insights into the molecular mechanisms modulating ACK1 and they position SIAH ubiquitin ligases as negative regulators of ACK1 in transformed cells.

MUC5B Leads to Aggressive Behavior of Breast Cancer MCF7 Cells

The mucin MUC5B has a critical protective function in the normal lung, salivary glands, esophagus, and gallbladder, and has been reported to be aberrantly expressed in breast cancer, the second leading cause of cancer-related deaths among women worldwide. To understand better the implication of MUC5B in cancer pathogenesis, the luminal human breast cancer cell line MCF7 was transfected with a vector encoding a recombinant mini-mucin MUC5B and was then infected with a virus to deliver a short hairpin RNA to knock down the mini-mucin. The proliferative and invasive properties in Matrigel of MCF7 subclones and subpopulations were evaluated in vitro. A xenograft model was established by subcutaneous inoculation of MCF7 clones and subpopulations in SCID mice. Tumor growth was measured, and the tumors and metastases were assessed by histological and immunological analysis. The mini-mucin MUC5B promoted MCF7 cell proliferation and invasion in vitro. The xenograft experiments demonstrated that the mini-mucin promoted tumor growth and MCF7 cell dissemination. In conclusion, MUC5B expression is associated with aggressive behavior of MCF7 breast cancer cells. This study suggests that MUC5B may represent a good target for slowing tumor growth and metastasis.

Associated expressions of FGFR-2 and FGFR-3: from mouse mammary gland physiology to human breast cancer

Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors which have been implicated in breast cancer. The aim of this study was to evaluate FGFR-1, -2, -3, and -4 protein expressions in normal murine mammary gland development, and in murine and human breast carcinomas. Using immunohistochemistry and Western blot, we report a hormonal regulation of FGFR during postnatal mammary gland development. Progestin treatment of adult virgin mammary glands resulted in changes in localization of FGFR-3 from the cytoplasm to the nucleus, while treatment with 17-β-estradiol induced changes in the expressions and/or localizations of FGFR-2 and -3. In murine mammary carcinomas showing different degrees of hormone dependence, we found progestin-induced increased expressions, mainly of FGFR-2 and -3. These receptors were constitutively activated in hormone-independent variants. We studied three luminal human breast cancer cell lines growing as xenografts, which particularly expressed FGFR-2 and -3, suggesting a correlation between hormonal status and FGFR expression. Most importantly, in breast cancer samples from 58 patients, we found a strong association (P<0.01; Spearman correlation) between FGFR-2 and -3 expressions and a weaker correlation of each receptor with estrogen receptor expression. FGFR-4 correlated with c-erbB2 over expression. We conclude that FGFR-2 and -3 may be mechanistically linked and can be potential targets for treatment of estrogen receptor-positive breast cancer patients.