[Rich66]

1: Cell Cycle. 2009 Oct 15;8(20):3297-302. Epub 2009 Oct 10. Links

Retinoid signaling regulates breast cancer stem cell differentiation.

Ginestier C, Wicinski J, Cervera N, Monville F, Finetti P, Bertucci F, Wicha MS, Birnbaum D, Charafe-Jauffret E.
Centre de Recherche en Cancérologie de Marseille, UMR891 Inserm/Institut Paoli-Calmettes, Marseilles, France. christophe.ginestier@inserm.fr
The cancer stem cell (CSC) hypothesis implicates the development of new therapeutic approaches to target the CSC population. Characterization of the pathways that regulate CSCs activity will facilitate the development of targeted therapies. We recently reported that the enzymatic activity of ALDH1, as measured by the ALDELFUOR assay, can be utilized to isolate normal and malignant breast stem cells in both primary tumors and cell lines. In this study, utilizing a tumorsphere assay, we have demonstrated the role of retinoid signaling in the regulation of breast CSCs self-renewal and differentiation. Utilizing the gene set enrichment analysis (GSEA) algorithm we identified gene sets and pathways associated with retinoid signaling. These pathways regulate breast CSCs biology and their inhibition may provide novel therapeutic approaches to target breast CSCs.
PMID: 19806016 [PubMed - in process

Anticancer Res. 2009 Dec;29(12):4959-4964.
Retinoid-induced Histone Deacetylation Inhibits Telomerase Activity in Estrogen Receptor-negative Breast Cancer Cells.

Phipps SM, Love WK, White T, Andrews LG, Tollefsbol TO.
Department of Biology, 175 Campbell Hall, 1300 University Boulevard, Birmingham, AL 35294-1170, U.S.A. trygve@uab.edu.
BACKGROUND: Multiple mechanisms regulate cancer-associated telomerase activity at the level of human telomerase reverse transcriptase (hTERT) transcription which may serve as novel targets for anticancer approaches. MATERIALS AND METHODS: The effects of prolonged all-trans retinoic acid (ATRA) exposure on hTERT regulation in estrogen receptor-negative SK-BR-3 breast cancer cells were examined. RESULTS: ATRA had a profound effect on the morphology and proliferation rate of the SK-BR-3 cells. ATRA also hindered the ability of these cancer cells to grow independently, rendering them more like normal somatic cells. The effect of ATRA on the decrease of telomerase activity was found to be associated with a rapid decrease in histone H3-lysine 9 acetylation (H3-K9-Ac) of the hTERT promoter. Extended-exposure to ATRA in these cells also caused the initiation of a putative compensatory mechanism, counteracting the induced surge in apoptosis. CONCLUSION: A rapid decrease of H3-K9 acetylation at the hTERT promoter could be an important mechanism by which ATRA shuts down telomerase activity and mediates its antitumor effects in estrogen receptor-negative breast cancer cells.

PMID: 20044602 [PubMed - as supplied by publisher]



Int J Mol Med. 2010 Feb;25(2):271-80.
Influence of LOX/COX inhibitors on cell differentiation induced by all-trans retinoic acid in neuroblastoma cell lines.

Redova M, Chlapek P, Loja T, Zitterbart K, Hermanova M, Sterba J, Veselska R.
Laboratory of Tumor Biology and Genetics, Institute of Experimental Biology, School of Science, Masaryk University, 61137 Brno, Czech Republic.
We investigated the possible modulation by LOX/ COX inhibitors of all-trans retinoic acid (ATRA)-induced cell differentiation in two established neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor of cyclooxygenase-2, were chosen for this study. The effects of the combined treatment with ATRA and LOX/COX inhibitors on neuroblastoma cells were studied using cell morphology assessment, detection of differentiation markers by immunoblotting, measurement of proliferation activity, and cell cycle analysis and apoptosis detection by flow cytometry. The results clearly demonstrated the potential of caffeic acid to enhance ATRA-induced cell differentiation, especially in the SK-N-BE(2) cell line, whereas application of celecoxib alone or with ATRA led predominantly to cytotoxic effects in both cell lines. Moreover, the higher sensitivity of the SK-N-BE(2) cell line to combined treatment with ATRA and LOX/COX inhibitors suggests that cancer stem cells are a main target for this therapeutic approach. Nevertheless, further detailed study of the phenomenon of enhanced cell differentiation by expression profiling is needed.

PMID: 20043138 [PubMed - in process]

1: Mol Cancer Ther. 2005 May;4(5):824-34. Links

The selective retinoid X receptor agonist bexarotene (LGD1069, Targretin) prevents and overcomes multidrug resistance in advanced breast carcinoma.

Yen WC, Lamph WW.
Department of Molecular Oncology, Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, CA 92121, USA.
Acquired drug resistance represents a major challenge in the therapeutic management of breast cancer patients. We reported previously that the retinoid X receptor-selective agonist bexarotene (LGD1069, Targretin) was efficacious in treating animal models of tamoxifen-resistant breast cancer. The goal of this study was to evaluate the effect of bexarotene on development of acquired drug resistance and its role in overcoming acquired drug resistance in advanced breast cancer. Paclitaxel, doxorubicin, and cisplatin were chosen as model compounds to determine the effect of bexarotene on the development of acquired drug resistance. Human breast cancer cells MDA-MB-231 were repeatedly treated in culture with a given therapeutic agent with or without bexarotene for 3 months. Thereafter, cells were isolated and characterized for their drug sensitivity. Compared with parental cells, cells treated with a single therapeutic agent became resistant to the therapeutic agent, whereas cells treated with the bexarotene combination remained chemosensitive. Cells with acquired drug resistance, when treated with the combination, showed increased sensitivity to the cytotoxic agent. Furthermore, cells treated with the combination regimen had reduced invasiveness and angiogenic potential than their resistant counterparts. These in vitro findings were further confirmed in an in vivo MDA-MB-231 xenograft model. Our results suggest a role for bexarotene in combination with chemotherapeutic agents in prevention and overcoming acquired drug resistance in advanced breast carcinoma.
PMID: 15897247 [PubMed - indexed for MEDLINE

http://breast-cancer-research.com/content/10/4/210


Breast cancer stem cells as therapeutic targets

In the past two decades, more than 30 new anticancer drugs have been introduced, but survival rates have improved only marginally for many forms of cancer [65]. In contrast to most cancer cells, cancer stem cells are slow-dividing and have a lowered ability to undergo apoptosis and a higher ability of DNA repair, making them more resistant to traditional methods of cancer treatment such as radiation and chemotherapy. In vitro experiments comparing differentiated breast cancer cells grown under monolayer conditions with CD24^-/low CD44⁺ cancer stem cells grown under mammosphere conditions showed that the stem cell-like population was more resistant to radiation [66]. In addition, stem cells express ABC drug transporters, which protect the cell from cytotoxic agents and may lead to MDR [67]. Current anti-cancer therapy is effective at debulking the tumour mass but treatment effects are transient, with tumour relapse and metastatic disease often occurring as a result of the failure of targeting cancer stem cells. For therapy to be more effective, debulking of differentiated tumours must occur followed by targeting of the remaining surviving, often quiescent, tumour stem cells. This could be accomplished by differentiating BCSCs through differentiating therapy or eliminating them via immunotherapy.
Differentiation therapy targeting cancer stem cells

One way to target cancer stem cells is to induce the cancer stem cells to differentiate. Targeting the cancer stem cell pool to differentiate results in the loss of the ability for self-renewal, a hallmark of the cancer stem cell phenotype and the reason behind maintenance of the cancer stem cells. One differentiation agent used in the clinic is retinoic acid (RA) (vitamin A) [68]. RA and vitamin A analogues can promote differentiation of epithelial cells and reverse tumour progression through modulation of signal transduction. RA-based therapy followed by chemotherapy has found use in acute promyeloctyic leukaemia and could also find use in solid tumour therapy [69]. Recently, the use of bone morpho-genetic protein (BMP)-4 has been described as a non-cytotoxic effector capable of blocking the tumourigenic potential of human glioblastoma cells [70]. This therapeutic agent is able to work by reducing proliferation and inducing expression of neural differentiation markers in stem-like tumour-initiating precursors. These findings are intriguing in light of the role that BMP-4 may play in some breast tumours [71]. Finding ways to specifically target BCSCs via differentiation therapy is an application that needs to be further defined.
Targeting stem cells for elimination

Much of cancer therapy research is focused on targeting specific markers on tumour cells that are overexpressed or mutated and that often represent essential genes/proteins or pathways thought to be important for the development of the tumour. For instance, traztuzamab (Herceptin^®) targets the HER-2/neu (ErbB2) oncogene, a member of the epidermal growth factor receptor (EGFR) kinase family, a protein overexpressed on roughly 30% of breast tumours [72]. While these approaches have seen some clinical successes, the cancer stem cell model predicts that only by targeting the remaining cells left over after treatment, the putative cancer stem cells, will significant clinical remissions of the disease occur. It is important to note not only that tumours may be driven by mutated proteins and inappropriate signalling, but also that epigenetic mechanisms of gene expression of genes involved in 'stem-ness' such as Oct4, Nanog, and Sox2 could be behind tumour formation [73]. Reversal of these epigenetic switches of cancer stem cells could be one novel way to target cancer stem cells. New therapeutics aimed at eliminating cancer stem cells could also be achieved through a variety of methods: targeting the self-renewal signalling pathways critical for cancer stem cells, targeting the ABC drug transporters that cancer stem cells use to evade chemotherapy, or inducing the immune system to eliminate the cancer stem cells through various immunotherapeutic interventions.
Targeting of molecular signalling pathways and drug transporters

The use of the steroid-like molecule cyclopamine to inhibit the Hh signalling pathway has shown some promise in inhibiting the growth of medulloblastoma and could be used in treatments of other tumours [68]. The Wnt pathway can also be inhibited through a variety of mechanisms. Targeting of β-catenin has received a lot of attention as RA has been shown to inhibit β-catenin activity [74] and tyrosine kinase inhibitors such as imatinib (Gleevac^®) have been shown to down-regulate β-catenin signalling [75]. Finally, the Notch pathway has also been investigated as a target. An antibody capable of blocking Notch-4 has been used ex vivo to block the formation of mammospheres from primary human specimens [76]. This indicates the potential to block the self-renewal capacity of BCSCs in the patient with this antibody and opens up the use of other antibody therapies in the elimination of BCSCs.
In vitro experiments have shown the resistance of BCSCs to chemotherapy and radiation. Recent clinical evidence has established that tumourigenic breast cancer cells with high expression of CD44 and low expression of CD24 are resistant to chemotherapy [77]. Breast cancer patients receiving neoadjuvant chemotherapy had an increase in the CD44⁺/CD24^low population of cells following treatment. These cells retained the capacity to form mammospheres (demonstrating self-renewal) and had an enhanced propensity for forming tumours in SCID/Beige mice compared with pretreatment samples, increasing from 4 of 14 (29%) to 7 of 14 (50%) patient samples transferred. Treatment of patients with HER-2-positive tumours with lapatinib, an EGFR and HER-2/neu (ErbB-2) dual-tyrosine kinase inhibitor, resulted in nonstatistically significant decreases in the percentage of CD44⁺/CD24^low population and in the ability for self-renewal as assessed by mammosphere formation. Thus, inhibition of regulatory pathways involved in self-renewal may confer improved clinical outcomes by targeting BCSCs.



Transl Oncol. 2010 Jun 1;3(3):149-52.
Loss of tumor-initiating cell activity in cyclophosphamide-treated breast xenografts.

Zielske SP, Spalding AC, Lawrence TS.

Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, USA
Address all correspondence to: Steven P. Zielske, PhD, 4310 Med Sci I, Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109. E-mail: szielske@med.umich.edu

FREE TEXT

Abstract

Cancer stem cells (CSCs) are a subpopulation of tumor cells with preferential tumor-initiating capacity and have been purported to be resistant to chemotherapy. It has been shown that breast CSC are, on average, enriched in patient tumors after combination neoadjuvant chemotherapy including docetaxel, doxorubicin, and cyclophosphamide (CPA). Here, we investigate the resistance of breast CSC to CPA alone in a xenograft model. CPA treatment led to a 48% reduction in tumor volume during a 2-week period. Cells bearing the CD44(+) CD24(-) phenotype were reduced by 90% (2.5% to 0.24%) in CPA-treated tumors, whereas cells with aldehyde dehydrogenase activity were reduced by 64% (4.7% to 1.7%). A subsequent functional analysis showed that CPA-treated tumors were impaired in their ability to form tumors, indicating loss of functional tumor-initiating activity. These results are consistent with a CSC phenotype that is sensitive to CPA and indicate that some patient CSC may not display the expected resistance to therapy. Deciphering the mechanism for this difference may lead to therapies to counteract resistance.

PMID: 20563255 [PubMed - in process]PMCID: PMC2887643Free PMC Article