Robert Suriano

Thyroid cancer is the most common endocrine-related cancer in the United States and its incidence is rising rapidly. Since among various genetic lesions identified in thyroid cancer, the BRAFV600E mutation is found in 50% of papillary thyroid cancers and 25% of anaplastic thyroid cancers, this mutation provides an opportunity for targeted drug therapy. Our laboratory evaluated cellular phenotypic effects in response to treatment with PLX4032, a BRAFV600E-specific inhibitor, in normal BRAF-wild-type thyroid cells and in BRAFV600E-positive papillary thyroid cancer cells.

Background: Rapid breast tumor development relies on formation of new vasculature to supply the growing malignancy with oxygenated blood. Previously we found that estrogen aided in this neovasculogenesis via recruitment of bone marrow derived endothelial progenitor cells (BM-EPCs), leading to increased vessel formation and vascular endothelial growth factor (VEGF) production in vivo. However, the cellular mechanism of this induction and the signaling pathways involved need elucidation. Results: Using the murine mammary cell line TG1-1 we observed estrogen (E₂) lead to an up regulation of hypoxia inducible factor-1 (HIF-1), an effect abrogated by the anti-estrogen Fulvestrant and the HIF-1 inhibitor YC-1 (3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole) suggesting the interchangeability of hypoxia and estrogen mediated effects. Estrogen modulation of HIF-1 and subsequent effects on endothelial cells is dependent on the Akt/PI3K pathway and protein synthesis as validated by the use of the inhibitors wortmannin and cycloheximide which abrogated estrogen’s effects respectively. Estrogen treated TG1-1 cells secreted higher levels of VEGF which were comparable to secreted levels from cells grown under hypoxic conditions. Soluble factors in conditioned media from E₂ treated breast cancer cells also lead to migration and tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Conclusions: Our data provide evidence that estrogen signaling mediates the tumor vasculogenic process required for breast cancer progression and involves a key regulator of the hypoxia signaling pathway. Further, hypoxia and estrogen are interchangeable as both similarly modulate epithelial-endothelial cell interaction.

Background: Thyroid cancer, as with other types of cancer, is dependent on angiogenesis for its continued growth and development. Interestingly, estrogen has been shown to contribute to thyroid cancer aggressiveness in vitro, which is in full support of the observed increased incidence of thyroid cancer in women over men. Provided that estrogen has been observed to contribute to increased angiogenesis of estrogen responsive breast cancer, it is conceivable to speculate that estrogen also contributes to angiogenesis of estrogen responsive thyroid cancer. Methods: In this study, three human thyroid cancer cells (B-CPAP, CGTH-W-1, ML-1) were treated with estrogen alone or estrogen and anti-estrogens (fulvestrant and 3,3′-diindolylmethane, a natural dietary compound) for 24 hours. The cell culture media was then added to human umbilical vein endothelial cell (HUVECs) and assayed for angiogenesis associated events. Vascular endothelial growth factor (VEGF) levels were also quantified in the conditioned media so as to evaluate if it is a key player involved in these observations. Results: Conditioned medium from estrogen treated thyroid cancer cells enhanced phenotypical changes (proliferation, migration and tubulogenesis) of endothelial cells typically observed during angiogenesis. These phenotypic changes observed in HUVECs were determined to be modulated by estrogen induced secretion of VEGF by the cancer cells. Lastly, we show that VEGF secretion was inhibited by the anti-estrogens, fulvestrant and 3,3′-diindolylmethane, which resulted in diminished angiogenesis associated events in HUVECs. Conclusion: Our data establishes estrogen as being a key regulator of VEGF secretion/expression in thyroid cells which enhances the process of angiogenesis in thyroid cancer. These findings also suggest the clinical utility of anti-estrogens as anti-angiogenic compounds to be used as a therapeutic means to treat thyroid cancer. We also observed that 3,3′-diindolylmethane is a promising naturally occurring anti-estrogen which can be used as a part of therapeutic regimen to treat thyroid cancer.

Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR) proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS), a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants. We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-κB nuclear translocation analyses in HEK-BLUE™-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant. Our data demonstrate the identification of synthetic peptides that mimic LPS by interacting with TLR-4. This LPS mimotope-TLR-4 interaction will allow for the development and use of these peptides as a new class of adjuvants, namely TLR-4 agonists.

Endothelial progenitor cells are increasingly being studied in various diseases ranging from ischemia, diabetic retinopathy, and in cancer. The discovery that these cells can be mobilized from their bone marrow niche to sites of inflammation and tumor to induce neovasculogenesis has afforded a novel opportunity to understand the tissue microenvironment and specific cell-cell interactive pathways. This review provides a comprehensive up-to-date understanding of the physiological function and therapeutic utility of these cells. The emphasis is on the systemic factors that modulate their differentiation/mobilization and survival and presents the challenges of its potential therapeutic clinical utility as a diagnostic and prognostic reagent.