Joanne T. Emerman

Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue.

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted, myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM), α6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18, keratin 19, EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of α6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors.

Comparison of free and liposome encapsulated doxorubicin tumor drug uptake and antitumor efficacy in the SC115 murine mammary tumor

Tumor drug uptake and antitumor efficacy of free and liposomal doxorubicin (DOX) were determined in the SC115 Shionogi mouse mammary tumor. Liposomal DOX systems were prepared by pH gradient-driven drug encapsulation in 170 nm egg phosphatidylcholine/cholesterol (55:45, mol ratio) vesicles. Intravenous injection of free DOX at 6.5 mg/kg, the maximum tolerated dose for free drug in the multiple dose therapy regimen, resulted in tumor-associated drug levels of 2.0 micrograms/g tissue at 1 h which remained constant over 24 h. Liposomal DOX injected at 6.5 mg/kg led to an accumulation of drug in the tumor from 2.6 micrograms/g tissue to 5.5 micrograms/g tissue between 1 h and 24 h, respectively. Increasing the dose of liposomal DOX to 13.0 mg/kg increased tumor drug uptake levels to 5.7 micrograms/g and 10.2 micrograms/g tissue at 1 h and 24 h, respectively. Administration of free or liposome encapsulated DOX every 7 days for 3 weeks resulted in a dose-dependent decrease in tumor growth rate. However, liposomal DOX injected at 6.5 mg/kg exhibited enhanced tumor growth inhibition compared to an equivalent dose of free drug. Further, the ability to administer increased doses of the less toxic liposomal DOX not only resulted in a greater inhibition of tumor growth but also significantly reduced tumor weight. Tumors weighing as much as 5 g were diminished to less than 0.5 g upon treatment with liposomal DOX at a dose of 13 mg/kg. In addition, groups receiving the highest liposomal DOX dose exhibited 25% complete tumor regression which persisted over the 50-day study period. These results demonstrate the ability of appropriately designed liposomal DOX systems to significantly enhance the delivery and retention of drug at solid tumor sites, resulting in increased therapeutic activity.

Celecoxib analogues disrupt Akt signaling, which is commonly activated in primary breast tumours

IntroductionPhosphorylated Akt (P-Akt) is an attractive molecular target because it contributes to the development of breast cancer and confers resistance to conventional therapies. Akt also serves as a signalling intermediate for receptors such as human epidermal growth factor receptor (HER)-2, which is overexpressed in 30% of breast cancers; therefore, inhibitors to this pathway are being sought. New celecoxib analogues reportedly inhibit P-Akt in prostate cancer cells. We therefore examined the potential of these compounds in the treatment of breast cancer. The analogues were characterized in MDA-MB-453 cells because they overexpress HER-2 and have very high levels of P-Akt.MethodsTo evaluate the effect of the celecoxib analogues, immunoblotting was used to identify changes in the phosphorylation of Akt and its downstream substrates glycogen synthase kinase (GSK) and 4E binding protein (4EBP-1). In vitro kinase assays were then used to assess the effect of the drugs on Akt activity. Cell death was evaluated by poly(ADP-ribose) polymerase cleavage, nucleosomal fragmentation and MTS assays. Finally, tumour tissue microarrays were screened for P-Akt and HER-2 expression.ResultsOSU-03012 and OSU-O3013 inhibited P-Akt and its downstream signalling through 4EBP-1 and GSK at concentrations well below that of celecoxib. Disruption of P-Akt was followed by induction of apoptosis and more than 90% cell death. We also noted that the cytotoxicity of the celecoxib analogues was not significantly affected by serum. In contrast, the presence of 5% serum protected cells from celecoxib induced death. Thus, the structural modification of the celecoxib analogues increased P-Akt inhibition and enhanced the bioavailability of the drugs in vitro. To assess how many patients may potentially benefit from such drugs we screened tumour tissue microarrays. P-Akt was highly activated in 58% (225/390) of cases, whereas it was only similarly expressed in 35% (9/26) of normal breast tissues. Furthermore, HER-2 positive tumours expressed high levels of P-Akt (P < 0.01), supporting in vitro signal transduction.ConclusionWe determined that Celecoxib analogues are potent inhibitors of P-Akt signalling and kill breast cancer cells that overexpress HER-2. We also defined an association between HER-2 and P-Akt in primary breast tissues, suggesting that these inhibitors may benefit patients in need of new treatment options.

Aldehyde Dehydrogenase Activity Is a Biomarker of Primitive Normal Human Mammary Luminal Cells

Elevated aldehyde dehydrogenase (ALDH) expression/activity has been identified as an important biomarker of primitive cells in various normal and malignant human tissues. Here we examined the level and type of ALDH expression and activity in different subsets of phenotypically and functionally defined normal human mammary cells. We find that the most primitive human mammary stem and progenitor cell types with bilineage differentiation potential show low ALDH activity but undergo a marked, selective, and transient upregulation of ALDH activity at the point of commitment to the luminal lineage. This mirrors a corresponding change in transcripts and protein levels of ALDH1A3, an enzyme involved in retinoic acid synthesis and the most highly expressed ALDH gene in normal human mammary tissue. In contrast, ALDH1A1 is expressed at low levels in all mammary epithelial cells. These findings raise interesting questions about the reported association of ALDH activity with breast cancer stem cells and breast cancer prognosis. STEM CELLS 2012; 30:344–348.

Psychosocial stressors and mammary tumor growth: an animal model.

Stressful life events and the ability to cope with stress may play a role in the progression of breast cancer; however, the complex relationship between stressors and tumor growth is difficult to investigate in humans. Our studies have utilized the androgen-responsive Shionogi mouse mammary carcinoma (AR SC115) in male mice to investigate the effects of social housing condition on tumor growth rates and responses to chemotherapy. We demonstrate that, depending on social housing condition, mammary tumor growth and response to chemotherapy can both increase and decrease. We have examined the possible role(s) of 1) psychosocial variables, 2) testosterone and corticosterone, hormones altered by stress and known to stimulate SC115 cells in vivo and in vitro, 3) NK cells, one of the bodys first lines of defense against tumor cells, 4) stress proteins, in mediating the differential tumor growth rates observed in our model. This review discusses the investigations we have undertaken to elucidate the mechanisms through which a psychosocial stressor, social housing condition, can alter tumor growth rate.

Mitogenic properties of insulin-like growth factors I and II, insulin-like growth factor binding protein-3 and epidermal growth factor on human breast stromal cells in primary culture.

Insulin-like growth factors I and II (IGF-I and IGF-II) are growth factors implicated in both normal mammary gland development and breast cancer. We have previously reported on the effects of components of the IGF system on breast epithelial cells. Since data suggests that stromal–epithelial interactions play a crucial role in breast cancer, we have now investigated the mitogenic properties of IGF-I, IGF-II, insulin-like growth factor binding protein-3 (IGFBP-3) and epidermal growth factor (EGF) on human breast stromal cells in primary culture. We show that, under serum-free conditions, stromal cells are stimulated to grow in response to IGF-I and IGF-II in a dose-dependent manner. IGF-I and EGF, a potent stimulator of human breast epithelial cell growth in primary culture and also associated with breast cancer, appear to stimulate stromal cell growth in a synergistic manner. IGFBP-3 does not inhibit the stimulation of growth by IGF-I, or IGF-I plus EGF. However, IGFBP-3 does inhibit the stimulation of growth by IGF-II. In contrast to our previous results with human breast epithelial cells, IGFBP-3 does not have an IGF-independent inhibitory effect on stromal cell growth. This study is the first to address the effects of IGF-I, IGF-II and IGFBP-3 alone and in combination with EGF on human breast stromal cell growth in primary culture. Characterizing the role of the IGF system in both normal breast epithelial cells and stromal cells will aid in our understanding of the mechanisms behind the role of the IGF system in breast cancer.

In vitro screening of crude extracts and pure metabolites obtained from marine invertebrates for the treatment of breast cancer.

SummaryA total of 15 samples (crude extracts and pure secondary metabolites) obtained from marine invertebrates collected from the offshore waters of British Columbia, Papua New Guinea, and Sri Lanka have previously been shown to exert cytotoxic activity in the in vitro L1210 leukemic bioassay. We screened these metabolites for in vitro cytotoxic activity against the drug-sensitive breast-tumor cell lines MCF-7, T-47D, ZR-75-1, and MDA-MB-231; the multidrug-resistant and P-glycoprotein (Pgp)-positive breast-tumor cell lines MCF-7 Adr and MDA-Alr; and normal and malignant human breast epithelial cells (HBEC) in primary culture. Eight samples exhibited significant [drug concentration resulting in a 50% decrease in cell growth as compared with controls (ED50),<25 μg/ml] dose-dependent cytotoxicity against the drug-sensitive cell lines; the ED50 values were as low as 0.004 μg/ml. Five of the eight samples exhibited significant cytotoxicity against the multidrug-resistant cell lines; the ED50 values were as low as 0.0006 μg/ml. Incubation of MCF-7 Adr cells with varying concentrations of compounds in the presence of Adriamycin demonstrated that none of the compounds tested interfered with Pgp function. Results obtained using HBEC in primary culture showed a wide range of chemosensitivities for a given drug against tissue taken from different patients, demonstrating the uniqueness of the response of different individuals to chemotherapy.

Effects of psychosocial stressors on mouse mammary tumor growth

We describe a sensitive and replicable animal model which demonstrates that psychosocial variables both increase and decrease tumor growth rate. The effects of social housing condition and daily exposure to novel environments were investigated in mice bearing the transplantable androgen-responsive Shionogi mouse mammary carcinoma (SC115). Being reared individually housed and remaining individually housed or being reared in a sibling group and then singly housed following tumor cell injection markedly increased tumor growth compared to that in mice remaining in their standard sibling rearing groups (n = 2-3 per group), if animals were also exposed to acute daily novelty stress. In contrast, being reared individually and then moved to a larger social group (n = 4-5 nonsiblings per group) markedly reduced tumor growth, both in the presence and absence of acute daily novelty stress.

The Luminal Progenitor Compartment of the Normal Human Mammary Gland Constitutes a Unique Site of Telomere Dysfunction

Telomeres are essential for genomic integrity, but little is known about their regulation in the normal human mammary gland. We now demonstrate that a phenotypically defined cell population enriched in luminal progenitors (LPs) is characterized by unusually short telomeres independently of donor age. Furthermore, we find that multiple DNA damage response proteins colocalize with telomeres in >95% of LPs but in <5% of basal cells. Paradoxically, 25% of LPs are still capable of exhibiting robust clonogenic activity in vitro. This may be partially explained by the elevated telomerase activity that was also seen only in LPs. Interestingly, this potential telomere salvage mechanism declines with age. Our findings thus reveal marked differences in the telomere biology of different subsets of primitive normal human mammary cells. The chronically dysfunctional telomeres unique to LPs have potentially important implications for normal mammary tissue homeostasis as well as the development of certain breast cancers.

Routine culturing of normal, dysplastic and malignant human mammary epithelial cells from small tissue samples

SummaryWe compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca++ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca++ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression.