Tamoxifen selects for breast cancer cells with mammosphere forming capacity and increased growth rate.

Using the M05 mouse mammary tumor model and the MCF-7 cell line, we investigated the effect of tamoxifen treatment on the fraction of breast cancer cells with self-renewing capacity both in vitro and in vivo. We found that pretreatment with 4-OH-tamoxifen leads to an increase in cells with the ability of forming mammospheres that express lower levels of ER-α and increased expression of transcription factors associated with pluripotency. Moreover, exposure on plastic to 4-OH-tamoxifen by itself leads to an upregulation of these transcription factors. M05 tumors grown in mice treated with tamoxifen have a higher percentage of cells with self-renewing capacity and this proportion is conserved when tumors are passaged to nontreated mice. Furthermore, interruption of tamoxifen leads to increased tumor growth compared to tumors grown in mice that were never exposed to the antiestrogen. In addition, these tumors are characterized by a higher number of CD24(l)CD29(h) cells compared to tumors grown in nontreated mice. Treatment in vitro with 4-OH-tamoxifen for 5 days leads to a long lasting increase in the proportion of cells with self-renewing capacity even after 1 month of growth in the absence of the antiestrogen. Finally, we compared the mammosphere forming capacity of hormone dependent and independent passages of the M05 tumor and found that hormone independence is associated to an increase in cells with self-renewing capacity. Our results support previous findings that suggest that endocrine treatment selects for cells with stem cell properties.

Is the epithelial-to-mesenchymal transition clinically relevant for the cancer patient?

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process by which a fully differentiated epithelial cell acquires mesenchymal traits, and therefore, mesenchymal abilities such as motility and invasiveness. It is a pivotal physiological process involved in embryogenesis (Type 1 EMT) and in wound healing and tissue remodeling (Type 2 EMT), which, some authors claim, but there are still some controversies, has also been co-opted by tumor cells to increase their malignant potential (Type 3 EMT). Many biomarkers of Type 3 EMT have been characterized and classified into functional categories (i.e., extracellular proteins, cell surface molecules, cytoskeletal markers, transcriptional factors, and, recently, micro RNAs). The extra and intracellular signals that lead to EMT are only starting to be understood, but there is a consensus that Ras and TGF-beta signaling must converge with NF-κB in order to achieve a full EMT. The most classical experimental model is the induction of EMT by TGF-beta in cultures of epithelial cells. Other pathways involving GSK3b, and Wnt/beta-catenin, are also implicated. Ultimately, every EMT-inducing pathway will activate any of the E-cadherin transcriptional repressors (ZEB1, ZEB2, Twist, Snail or Slug). Although in the pre-clinical setting, EMT has also been related to an accelerated tumor progression and to an increased resistance to conventional chemotherapy. In this sense, several groups are beginning to use EMT as a predictive marker of response to treatment. Finally, two chemicals targeting TGF-beta are in clinical trials and many laboratories have initiated studies to use other EMT-related molecules as a therapeutic target for the cancer patient with some modest, but encouraging results.

A spontaneous estrogen dependent, tamoxifen sensitive mouse mammary tumor: a new model system to study hormone-responsiveness in immune competent mice.

Currently, an in vivo spontaneous model of estrogen dependent, tamoxifen sensitive breast cancer does not exist. We show here the characterization of the M05 mammary tumor that appeared spontaneously in a 1-year-old virgin female BALB/c mouse in our animal facility. The M05 tumor is a semi-differentiated adenocarcinoma that expresses estrogen and progesterone receptors. When it was transplanted to either male or ovariectomized female mice it did not grow. Moreover, ovariectomy or treatment with tamoxifen of tumor bearing mice led to a halt in tumor growth. Treatment of ovariectomized mice that had been inoculated with the M05 tumor showed that only estradiol, but not progesterone, promoted the re-growth of the tumor. Finally, after passage nine, tumor growth was achieved in male and ovariectomized female mice suggesting that the tumor had progressed to hormone independence. However, like often found in the clinic, expression of estrogen and progesterone receptors was maintained. This model mimics the biology of estrogen receptor positive breast cancer in humans and presents itself as an invaluable tool for the study of endocrine resistance in a physiologically relevant setting.

Establishment of an in vitro estrogen-dependent mouse mammary tumor model: a new tool to understand estrogen responsiveness and development of tamoxifen resistance in the context of stromal-epithelial interactions.

Currently, to our knowledge, there are no continuous cell lines derived from estrogen dependent, tamoxifen sensitive spontaneous mouse mammary carcinomas. We describe here the establishment and characterization of a cell line derived from the M05 mouse mammary tumor, LM05-Mix, composed of both an epithelial and a fibroblastic component. From it the respective epithelial LM05-E and fibroblastic LM05-F cell lines were generated by limiting dilution. Immunofluorescence studies confirmed that the epithelial cells were positive for E-cadherin, cytokeratins and vimentin whereas the fibroblastic cells were negative for the epithelial markers and positive for alpha-smooth muscle actin and vimentin. Both cell types expressed estrogen and progesterone receptors, although only the epithelial LM05-E cells were stimulated by estradiol and inhibited by tamoxifen. In the bicellular LM05-Mix cell line estradiol proved to stimulate cell proliferation whereas the response to tamoxifen was dependent on confluency and the degree of epithelial-fibroblastic interactions. The presence of membrane estrogen receptors in both cell types was suggested by the achievement of non-genomic responses to short treatments with estradiol, leading to the phosphorylation of ERK1/2. Finally, cytogenetic studies suggest that these two cell types represent independent cell populations within the tumor and would not be the result of an epithelial-mesenchymal transition. This model presents itself as a valuable alternative for the study of estrogen responsiveness and tamoxifen resistance in the context of epithelial-stromal interactions.

Expression pattern of glypican-3 (GPC3) during human embryonic and fetal development.

Glypicans represent a family of cell surface proteoglycans. Loss-of-function mutations in the human glypican-3 (GPC3) gene results in the Simpson-Golabi-Behmel syndrome, characterized by severe malformations and pre- and postnatal overgrowth. Because the expression of GPC3 during human embryonic and fetal periods remains largely unknown, we investigated by immunohistochemistry its pattern of expression during four periods of human development covering the embryonic period (P1) from 5 to 8 weeks of development, and the fetal periods (P2, P3 and P4) from 9 to 28 weeks of development. Hepatocytes were homogeneously positive for GPC3 during the four periods while pancreatic acini and ducts showed a rather high staining only during P1. GPC3 was also detected in several kidney structures and in the genital system where the sex cords were weakly positive in P1 and P2. In later developmental stages the male's genital system expressed GPC3 while the female's did not. While the mesenchyme in the limbs showed positive staining in P1, GPC3 was not detected during the following stages. The mesenchymal tissue localized between the most caudal vertebrae was also positive in P1. A strong GPC3 signal was observed in neurons of the spinal cord and dorsal root ganglia in P2 and P3, while the brain was negative. In sum our studies revealed that GPC3 expression is highly tissue- and stage-specific during human development. The expression pattern of GPC3 is consistent with the abnormalities seen in the Simpson-Golabi-Behmel syndrome.

Prognostic value of E-cadherin, beta-catenin, MMPs (7 and 9), and TIMPs (1 and 2) in patients with colorectal carcinoma.

BACKGROUND AND OBJECTIVES: Therapy of colorectal tumors (CRC) based on histology and clinical factors is insufficient to predict the evolution of each patient. The finding of molecular abnormalities able to differentiate subgroups of patients with bad prognosis will improve our ability to treat them successfully. Our purpose was to analyze retrospectively the prognostic input of E-cadherin, beta-catenin, metalloproteinases (MMPs) (7 and 9), and tissue inhibitors of metalloproteinases (TIMPs) (1 and 2) in patients with a follow-up period of 5 years. METHODS: Antigen expression was analyzed by immunohistochemistry. Prognostic evaluation was performed with the multivariate proportional hazards model. RESULTS: We demonstrated a concomitant loss of E-cadherin and beta-catenin at membranous level and an abnormal accumulation of nuclear beta-catenin. Besides, we found that all MMPs and TIMPs studied were overexpressed in CRC tissue. There was no association between the expression of any of these molecules and the known clinical-pathological parameters employed in CRC pathology. A multivariate analysis demonstrated that the overall survival could be independently predicted by the loss of E-cadherin and the overexpression of TIMP-2. CONCLUSIONS: The expression of E-cadherin and TIMP-2 could be relevant in determining the prognosis of CRC patients and providing a more accurate mechanism for their classification.

EGF-R and PDGF-R, but not bcl-2, overexpression predict overall survival in patients with low-grade astrocytomas.

BACKGROUND AND OBJECTIVES: Therapy of malignant glioma tumors is based on histology and clinical factors. However, comparable lesions may correspond with important prognostic differences. Our purpose was to analyze retrospectively the prognostic input of platelet-derived growth factor receptor (PDGF-R), epidermal growth factor (EGF-R), and bcl-2 expression in 103 malignant gliomas from uniformly treated patients. METHODS: The expression of the antigens was analyzed by immunohistochemistry (IHC). Prognostic evaluation was performed with the multivariate proportional hazards model. The follow-up period lasted 19 (5-122) months for survivors. RESULTS: We observed that almost 50% of gliomas showed high expression of PDGF-R, while a lower expression of EGF-R and bcl-2 was found. No association between the main prognostic factors in malignant glioma (sex, age, histological grade, and Karnofsky score) and the labeling index (LI) of these antigens was observed. We found that only PDGF-R and EGF-R overexpression were associated with a shorter survival in patients with World Health Organization (WHO) II astrocytomas, being both associations independent of known prognostic factors, as shown by Cox model. Besides, we confirmed other authors' results that high histological grade and low performance score were associated with worse prognosis. CONCLUSIONS: PDGF-R and EGF-R expression could be relevant in determining the prognosis of low-grade astrocytomas (LGAs) and in providing a more objective mechanism for their classification.

Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum.

Meridianins are brominated 3-(2-aminopyrimidine)-indoles which are purified from Aplidium meridianum, an Ascidian from the South Atlantic (South Georgia Islands). We here show that meridianins inhibit various protein kinases such as cyclin-dependent kinases, glycogen synthase kinase-3, cyclic nucleotide-dependent kinases and casein kinase 1. Meridianins prevent cell proliferation and induce apoptosis, a demonstration of their ability to enter cells and to interfere with the activity of kinases important for cell division and cell death. These results suggest that meridianins constitute a promising scaffold from which more potent and selective protein kinase inhibitors could be designed.