Faculty Bibliography

Estrogen receptors (ER) play important roles in the development and progression of breast and ovarian cancers. ERs mediate transcriptional regulation through interaction with cofactors and binding to response elements within the regulatory elements of target genes. Here, we examined the expression and function of TBLR1/TBL1XR1, a core component of NCoR (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid receptor) corepressor complexes, in breast and ovarian cancers. We found that although TBLR1 is present in both the nucleus and cytoplasm of normal and neoplastic breast and ovarian cells, it is expressed at significantly higher levels in the nucleus of malignant breast and ovarian cells compared to benign cells. TBLR1 functions as an ER corepressor to inhibit ER-mediated transcriptional activation in both breast and ovarian cell lines, but it has no effect on androgen receptor (AR) mediated transcriptional activation in these cells. Furthermore, ectopic expression of nuclear TBLR1 in breast and ovarian cancer cells stimulates cell proliferation. The increased cell proliferation by nuclear TBLR1 is through both ER-independent and ER-dependent mechanisms as evidenced by increased growth in hormone-free medium and estrogen medium, as well as reduced growth with ER knockdown by siRNA. Nuclear TBLR1 overexpression also increased migration and invasion in both breast and ovarian cancer cells. Determining the functional relationship between TBLR1 and ER may provide insights to develop novel treatment strategies and improve response to hormonal therapy in breast and ovarian cancers.

Abstract Objective: The placenta of mid-gestation mice is a known rich source of hematopoietic stem cells. We hypothesized that it is also a source of other multipotent stem cells. Methods: We isolated fetal cells from the murine placenta across the second half of gestation and characterized their expression of surface antigens known to be associated with mesenchymal stem cells (MSCs) on a subset of hematopoietic lineage-negative cells. Using real-time reverse-transcriptase quantitative polymerase chain reaction, we also evaluated the expression of intracellular transcription factors (TFs) known to be associated with renal development and/or multipotent stem cells. Results: Cell phenotypes with surface marker and TF expression consistent with multipotent stem cells of a mesenchymal lineage as well as renal cell progenitors were found in the placenta. The expression of MSC and renal progenitor surface markers varied throughout gestation, but was highest on E12-15 where such cells represented a small but significant percentage of the population. Of the studied TFs, 10 of 11 renal TFs were found at moderate to high levels, and all stem cell TFs were found. Conclusion: The mid-gestation murine placenta may serve as a source of multipotent stem cells and also contains cells which may be renal cell progenitors.

Androgen receptor (AR), a steroid hormone receptor, is critical for prostate cancer growth. However, activation of AR by androgens can also lead to growth suppression and differentiation. Transcriptional cofactors play an important role in this switch between proliferative and anti-proliferative AR target gene programs. Transducin beta-like-related protein 1 (TBLR1), a core component of the nuclear receptor corepressor complex, shows both corepressor and coactivator activities on nuclear receptors, but little is known about its effects on AR and prostate cancer. We characterized TBLR1 as a coactivator of AR in prostate cancer cells and determined that the activation is dependent on both phosphorylation and 19S proteosome. We showed that TBLR1 physically interacts with AR and directly occupies the androgen-response elements of the affected AR target genes in an androgen-dependent manner. TBLR1 is primarily localized in the nucleus in benign prostate cells and nuclear expression is significantly reduced in prostate cancer cells in culture. Similarly, in human tumor samples, the expression of TBLR1 in the nucleus is significantly reduced in the malignant glands compared with the surrounding benign prostatic glands (P<0.005). Stable ectopic expression of nuclear TBLR1 leads to androgen-dependent growth suppression of prostate cancer cells in vitro and in vivo by selective activation of androgen-regulated genes associated with differentiation (e.g. KRT18) and growth suppression (e.g. NKX3-1), but not cell proliferation of the prostate cancer. Understanding the molecular switches involved in the transition from AR-dependent growth promotion to AR-dependent growth suppression will lead to more successful treatments for prostate cancer.

Amniotic fluid contains a mixture of cells with capacity to differentiate into all germ layers. These cells are present in large numbers in midtrimester samples obtained for cytogenetic diagnosis, and have been identified by stem cell surface markers and transcription factors. We studied cultured samples from patients who had both direct cultures and matched cultures obtained 2 weeks later from the cytogenetics laboratory as well as patients with cytogenetics material only. Samples were cryogenically frozen, thawed, expanded in culture with excellent viability. There was considerable individual variation unrelated to gestational age or telomere length. Phenotype for embryonic markers was assessed by flow cytometry and by quantitative polymerase chain reaction. The most consistently present stem cell markers in substantial amounts were CD90, SSEA-4, & TRA-1-60. Cells with CD90, SSEA-4 & TRA-1-60 double and triple labeled also could be identified and subcultured, confirming the heterogeneity of the amniotic fluid stem cell population

OBJECTIVE: We sought to characterize markers of pluripotency in amniotic fluid derived cells as a non-controversial and readily available source of potentially therapeutic stem cells. STUDY DESIGN: Amniotic fluid stem cells (AFSC) express stem cell surface markers as well as transcription factors. Isolation and enrichment of the stem cell population is essential to their therapeutic potential. We studied expression of stem cell surface markers CD 117, CD 133, SSEA3, SSEA4, TRA 160, TRA 181 and CD90; as well as transcription factors OCT4, SOX2, NANOG and REX 1 by magnetic bead separation, flow cytometry analysis and PCR for transcription factors. Samples were obtained after cytogenetic analysis following routine amniocentesis for age or maternal anxiety in 10 normal patients. Cells were cultured for up to seven passages with analysis after confluence. RESULTS: There was great variation in different samples ability to express the different markers. The most prevalent marker was CD90, a mesenchymal stem cell factor; followed by SSEA4 and TRA160, both embryonic stem cell markers. The other markers were significantly present as well (SSEA3, TRA 160, TRA 181,CD90,OCT4, SOX2, NANOG and REX 1), however CD 117 and CD 133 were often undetectable or present in small amounts. CONCLUSION: There was considerable variation among samples, possibly gestational age related. In Amniotic fluid derived cells, stem cell markers CD90, SSEA4 and TRA160 are expressed in the largest amount with CD90 being the most abundantly expressed marker. Therefore these markers might be used for identification, isolation and enrichment of amniotic fluid stem cells for potential clinical use, since amniotic fluid is widely available and non-controversial as a source of multipotent stem cells.(Table persented)

AIM: To evaluate circulating endothelial lineage cells (ELCs) as biomarkers of tumor neovascularization in patients with pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: ELCs were isolated from the peripheral blood of patients with PDAC (n=14) or controls (n=17) before and after tumor resection/surgery and quantified using flow cytometry. Vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) were detected in tumor using immunohistochemistry and in plasma using an ELISA technique. RESULTS: Circulating ELC levels were increased in patients with PDAC compared to controls. After PDAC resection, ELC levels declined. ELC level increases were associated with cancer recurrence. VEGF and PlGF were identified in cancer cells and exocrine pancreas cells. Only PlGF was detected in tumor-associated inflammatory cells. Plasma levels of PlGF were higher in patients with PDAC compared to controls. CONCLUSION: Circulating ELCs are a potential biomarker of PDAC neovascularization, and PlGF may be an important target in treatment of PDAC