Throughout the years a number of scientists have come from around the world to work in the Breast Cancer Research Laboratory (BCRL) in order to complete their training, as a part of a rotation within their PhD program, or as a visiting scholar on sabbatical from their University.
Stefanie Raymond-Whish, a PhD Candidate from the Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona came as a visiting Scholar in January 2007 to learn under Dr. Irma H. Russo the Morphometric Analysis of Mammary Gland development.
Daniel Tiezzi, MD, PhD, from the Hospital das Clinicas FMRPUSP, Department de Ginecologia
e Obstetricia, Ribeirao.Preto, Sao Paulo, Brasil.
Dr. Tiezzi was a Visiting Scholar supported by CAPES (Coordenação de Aperfeicoamento de Perssoal de Nivel Superior) of Brazil. During his stay (from March 2006 to February 2007) he worked in collaboration with Dr. Sandra Fernandez to understand the process of epithelial-mesenchymal transition (EMT) in epithelial cells, a phenomena that has been indicated as an important component of neoplastic transformation. His work aimed to elucidate the genetic mechanism involved in this process. He evaluated the expression of different genes related to EMT such as E-cadherin, TGFß1, TGFß2, h-RAS, TWIST1, SNAIL2, SMAD5, FN1, CEACAM1 and JAG1 using the in vitro- in vivo model of the estrogen induced cell transformation developed in our laboratory (Link to Research Projects: Estrogens and Breast Cancer). In this model the E2-transformed MCF-10F (E2 70) cells and the tumorigenic cell line C5-A8-T8 (C5-T8) exhibit progressive loss of ductulogenesis as demonstrated by growth in collagen matrix. MCF-10F cells form ductal structures while E2 70 cells form solid spherical masses that in histological sections exhibit a pattern of growth resembling ductal hyperplasia or carcinoma in situ. The tumorigenic cells C5-T8 did not form structures on collagen acquiring an invasive pattern with spindle like features. Dr Tiezzi and Fernandez observed a reduction in E-cadherin expression in E2 70 cells and a completely lost in C5-T8 cells. TGFß1, TGFß2, CEACAM1 and JAG1 were down regulated in E2 70 and C5-T8 cells. SMAD5 and h-RAS were up regulated in the tumorigenic C5-T8 cells whereas FN1, Twist1 and Snail2 were up regulated in C5-T8 and down regulated in E2 70.
The work performed by Drs. Tiezzi and Fernandez concluded that theloss of expression of TGFß1, TGFß2, CEACAM1 and JAG1 are related to ductulogenesis and branching and the overexpression of h-RAS with loss of E-cadherin expression and up modulation of TWIST1, SNAIL2 and SMAD5 expressions involved in the EMT modulation.
Hilal Kocdor, MD, PhD, from the Institute of Oncology at the Dokuz Eylul University in Inciralti, Turkey was a Visiting Research Scientist to the BCRL at the Fox Chase Cancer Center from September 2007 to June 2008.
During her stay she studied in collaboration with Drs. M.A. Kocdor, Sandra Fernandez, Johana Vanegas and Irma H, Russo, the preventive effect of Human chorionic Gonadotropin (r-hCG) in the Neoplastic Transformation of Human Breast Epithelial Cells by 17 Beta Estradiol that was presented in the International Conference on Gonadotropins & Receptors, Hartfordshire, UK July 6-9, 2008. This work was based on the fact that structural alterations on ductal morphology are being considered early hallmarks of neoplastic transformation. Our previous data [See in Research Projects: Prevention Trial in the use of r-hCG in Women of High Risk of Developing Breast Cancer] have shown that, similar to early full-term pregnancy, r-hCG is capable of altering epithelial differentiation and able to elicit lifetime refractoriness to estrogen-induced neoplastic transformation on breast epithelium. In the present work we have developed a quantitative method that allows statistical comparisons for the evaluation of morphogenesis and phenotypic behaviors of transformed and non-transformed mammary epithelial cells (MCF-10F) in 3D collagen matrix which recapitulates in vivo development, and demonstrate the direct effect of r-hCG on the transformation phenotype.
To studyi the effect of r-hCG on the transformation of MCF-10F cells induced by 17 b-estradiol (E2), the cells were cultured for two weeks in the presence of 70nM E2 alone, or in combination with 50IU/ml rhCG. As a control, MCF-10F were treated with DMSO (solvent in which E2 was dissolved) or rhCG alone. At the end of the treatment, the cells were plated in collagen matrix (3-D culture) for eight days and 20 replicas were used for evaluating the length and the diameters of the main ducts, the diameter of solid masses, and the number of primary, secondary and tertiary branching arising from main ductules. Ductal structures obtained from collagen sections were also evaluated histologically. Analysis of the variance (ANOVA) followed by Tukey’s test was used for the comparisons of quantitative parameters.
E2 significantly increased solid mass formation (both in number and size) (p<0,001) and main ductal width (p<0,001) when compared to other groups with no effect on the ductal length and branching. E2 also caused remarkable disruption of monolayer luminal architectures. Total numbers of ductules were found to be similar between the treatment groups. However, the number of intermediate forms in E2+hCG group was significantly higher than the others (p<0,001). The formation of primary and secondary branching were higher for the E2+hCG group than control and DMSO (p=0,005; p=0,010). Tertiary branching was observed only in hCG-treated groups (hCG and hCG+E2). In addition, the number of tertiary branches in hCG group was higher than combination (Student’s t test, p<0,001). Main ductal lengths in hCG-treated groups (hCG and hCG+E2) were significantly longer than the others (p<0,001).
E2-induced neoplastic epithelial transformation is associated with impaired ductal morphogenesis in 3D culture. hCG has direct effects on branching morphogenesis and plasticity of breast epithelium. The loss of differentiation and disorganized growth of transformed MCF10F cells caused by estrogens is preventable by hCG-treatment.
Mehmet Ali Kocdor, MD, from the Department of Surgery of the Dokuz Eylul University Hospital at Inciraiti, Izmir, Turkey came to the BCRL on a Fulbright Scholarship [Insert link with the Fulbright] from September 2007 to June 1, 2008.
During his stay he studied in collaboration with dr. Johana Vanegas, Julia Pereira, Dr. Hilal Kocdor, Dr. Irma H. Russo, Sr. Fathima Sheriff, Kara Snider, and Dr. Jose Russo, “The preventive effect of hCG on rat mammary carcinogenesis”, that was presented at the International Conference on Gonadotropins & Receptors, Hartfordshire, UK July 6-9, 2008. This work was based on epidemiological evidence indicating that women’s risk of developing breast cancer is decreased by a full-term pregnancy at a young age. [Link to Research Projects: Prevention Trial in the use of r-hCG in Women of High Risk of Developing Breast Cancer]. Previous in vitro and in vivo work in our laboratory has shown that treatment of young virgin rats with hCG, like full-term pregnancy, efficiently inhibits the initiation and progression of chemically induced mammary carcinomas. Because this hormone could be used as a preventive agent to reduce women’s risk of developing breast cancer we tested the preventive effect of three different lengths of hCG treatment in a chemically induced model of mammary carcinogenesis. For this purpose, 36 day- old Sprague Dawley virgin rats were divided into 4 groups, and acclimatized to standard laboratory animal facility conditions for one week. At 50 days of age the rats were treated intra-peritoneally with 100IU (5ug) of hCG, for five, ten and fifteen consecutive days. The control group received for fifteen days the hormone vehicle only. When the rats reached 86 days of age, mammary carcinogenesis was induced with a single dose of 80mg/Kg of body weigh of DMBA, administered intra-gastrically. The animals were manually palpated and weighed each week. The incidence, number, size and location of tumors were recorded. Around 120 days after DMBA administration the animals were sacrificed, mammary tumors were excised, measured and were collected for histological and genomic analysis. Tumors were evaluated by their histological characteristics, location, quantity and size. There was a decrease in tumor incidence and a decreased number of tumors per animal which were directly related to the length of treatment. The control group had 90.9% of mammary cancer incidence, while for the hCG treated groups it was: 69.2%, 50%, 15.4% respectively for the 5, 10 and 15 days of hormonal treatment. All tumors were adenocarcinomas. A t-test showed significant difference between the control group and the hCG treated ones (p<0.03), as well as a statistical significance between 5 and 15 days of hCG (p<0.04). The tumor load per rat (mean ± SD) for the control group was 4.5±1.4, while for the hCG treated groups it was: 1.2±0.3, 1.4±0.5 and 0.4±0.2 for the 5, 10 and 15 days treatment respectively. A survival curve (Kaplan Meier) demonstrated that the percentage of animals free of tumors decrease significantly, accordingly with the decrease length of hCG treatment (Log rank p=0.0002). Animals treated with hCG also exhibited a increased latency in addition to the reduced incidence and multiplicity of DMBA-induced mammary adenocarcinomas.
These findings are of great interest to public health because they demonstrate preventive effect from cancer initiation even with a short length of hCG treatment, therefore offering novel approaches for the prevention of breast cancer.
Pedro J. Gutierrez, PhD, Associate Professor, Dpto. de Fundamentos del Analisis Economico e Historia e Instituciones Economicas, Facultad de Ciencias Economicas y Empresariales Universidad de Valladolid, Valladolid, Spain.
Dr. Gutierrez was a Visiting Scholar in the BCRL for the month of December 2007.
During his stay he established a collaborative work with Dr. Irma H. Russo to develop new methods for the mathematical formulation of cancer. They used the Optimal Control Theory, mainly used in Economics, and applied it to the analysis of biological behaviorsand illustrating the ability of this mathematical branch to describe biological phenomena and biological interrelationships.
Victoria Wargon, MS, from the Instituto de Biologia y Medicina Experimental (IByME), Buenos Aires, Argentina was a Visiting Scholar under a fellowship from the International Union Against Cancer from April to May 2008.
This fellowship was based on the finding by Wargon that acquired and constitutive antiprogestin resistance correlates with a diminished expression of Progesterone Receptor A (PR-A), suggesting that PR A may be regulated by epigenetic mechanisms. During her stay in the BCRL she was able to demonstrate, under the guidance of Dr. S. Fernandez, that the promoters or PR-A are methylated in mammary carcinomas with constitutive antiprogestin resistance but not in those with acquired resistance.