Robert Bednarczyk

mTOR inhibitors sensitize thyroid cancer cells to cytotoxic effect of vemurafenib.

Treatment options for advanced metastatic thyroid cancer patients are limited. Vemurafenib, a BRAFV600E inhibitor, has shown promise in clinical trials although cellular resistance occurs. Combination therapy that includes BRAFV600E inhibition and avoids resistance is a clinical need. We used an in vitro model to examine combination treatment with vemurafenib and mammalian target of rapamycin (mTOR) inhibitors, metformin and rapamycin. Cellular viability and apoptosis were analyzed in thyroid cell lines by trypan blue exclusion and TUNEL assays. Combination of vemurafenib and metformin decreased cell viability and increased apoptosis in both BCPAP papillary thyroid cancer cells and 8505c anaplastic thyroid cancer cells. This combination was also found to be active in vemurafenib-resistant BCPAP cells. Changes in expression of signaling molecules such as decreased mTOR expression in BCPAP and enhanced inhibition of phospho-MAPK in resistant BCPAP and 8505c were observed. The second combination of vemurafenib and rapamycin amplified cell death in BCPAP cells. We conclude that combination of BRAFV600E and mTOR inhibition forms the basis of a treatment regimen that should be further investigated in in vivo model systems. Metformin or rapamycin adjuvant treatment may provide clinical benefits with minimal side effects to BRAFV600E-positive advanced thyroid cancer patients treated with vemurafenib.

Discovery of a Novel Toxoplasma gondii Conoid-Associated Protein Important for Parasite Resistance to Reactive Nitrogen Intermediates

Toxoplasma gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors, including NO. Mechanisms used by T. gondii to modulate activation of its infected host cell likely underlie its ability to hijack monocytes and dendritic cells during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation, we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naive macrophages. We identified 14 independent T. gondii insertional mutants out of >8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild-type parasites recapitulated the RNI-resistance defect in the counter-immune mutant, confirming the role of the protein in resistance to macrophage RNIs.

Disruption of mutated BRAF signaling modulates thyroid cancer phenotype.

BackgroundThyroid 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.MethodsNormal BRAF-wild-type thyroid cells and BRAFV600E-mutated papillary thyroid cancer cells were subjected to proliferation assays and analyzed for cell death by immunofluorescence. Cell cycle status was determined using an EdU uptake assay followed by laser scanning cytometry. In addition, expression of proteins within the MAPK signal transduction pathway was analyzed by Western blot.ResultsPLX4032 has potent anti-proliferative effects selectively in BRAF-mutated thyroid cancer cells. These effects appear to be mediated by the drug’s activity of inhibiting phosphorylation of signaling molecules downstream of BRAF within the pro-survival MAPK pathway. Interestingly, PLX4032 promotes the phosphorylation of these signaling molecules in BRAF-wild-type thyroid cells.ConclusionsThese findings support further evaluation of combinational therapy that includes BRAFV600E inhibitors in thyroid cancer patients harboring the BRAFV600E mutation.

Abstract 1432: CXCL8 is a secretory inflammatory stimulus of the activated TME that modulates breast cancer phenotype.

The tumor microenvironment (TME) is a heterogeneous niche consisting of numerous stromal cells, structural components, and secreted molecules from both the malignant and surrounding cells that contribute to tumor growth and progression. Importantly, these diverse types of secreted molecules can initiate crosstalk events between the tumor cells and TME. One such molecule, CXCL8/Interleukin-8 (IL-8), is a proinflammatory chemokine, primarily involved in neutrophil mobilization, which activates multiple cellular signaling pathways via the membrane associated G protein-coupled CXCL8 cognate receptors, CXCR1 and CXCR2. We hypothesized that IL-8, a ubiquitous inflammatory molecule that is secreted by macrophages, activated fibroblasts and mesenchymal cells, can alter breast cancer phenotype. To this end, using a cell culture model we analyzed both estrogen receptor (ER) positive and ER negative breast cancer cell lines T47D and MDA-MB-231 respectively that were found to express the CXCL8 receptors CXCR1 and CXCR2. The cellular source of IL-8 was from conditioned media (CM) from in vitro cultures of primary stromal cells including primary mesenchymal and fibroblast cells, as well as the established monocytic cell lines HL-60 and THP-1. Each cell line secreted adequate amounts of IL-8 as measured by ELISA, however primary mesenchymal cells produced four times that of the fibroblasts and macrophages suggesting that activated mesenchymal cells may be a very potent source of chronic inflammation in the TME. Interestingly, treatment with IL-8 or CM from the phorbol ester treated macrophages THP-1 led to increased proliferation in T47D in vitro however only IL-8 treatment led to proliferation of MDA-MB-231. Recombinant IL-8 as well as CM of THP-1 and mesenchymal cells enhanced migration and invasion of both T47D and MDA-MB-231 cells as evaluated by a modified Boyden chamber assay. Supplementation of the triple negative MDA-MB-231 line with THP-1 CM enhanced its invasive propensity nearly two fold. THP-1 CM treatment also led to the activation of the PI3K/p-Akt and Mek/Erk pathways in the ER− MDA-MB-231 cells but not the ER+ T47D cells suggesting a link between CXCL8 mediated inflammation and constitutive signal transduction pathways that is translatable into enhanced metastatic propensity. Our studies are directed towards the analysis of both chronic and acute inflammation of the TME and we believe IL-8 and its associated intracellular molecular markers may be important preventive and therapeutic targets in triple negative breast cancer. Citation Format: Andrea L. George, Robert Bednarczyk, Shilpi Rajoria, Elyse Hanly, Robert Suriano, Abraham Mittelman, Raj K. Tiwari. CXCL8 is a secretory inflammatory stimulus of the activated TME that modulates breast cancer phenotype. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1432. doi:10.1158/1538-7445.AM2013-1432

Macrophage inflammatory factors promote epithelial-mesenchymal transition in breast cancer

The majority of breast cancers (90–95%) arise due to mediators distinct from inherited genetic mutations. One major mediator of breast cancer involves chronic inflammation. M1 macrophages are an integral component of chronic inflammation and the breast cancer tumor microenvironment (TME). Previous studies have demonstrated that up to 50% of the breast tumor comprise of tumor-associated macrophages (TAMs) and increased TAM infiltration has been associated with poor patient prognosis. Furthermore, breast cancer associated deaths are predominantly attributed to invasive cancers and metastasis with epithelial-mesenchymal transition (EMT) being implicated. In this study, we investigated the effects of cellular crosstalk between TAMs and breast cancer using an in vitro model system. M1 polarized THP-1 macrophage conditioned media (CM) was generated and used to evaluate cellular and functional changes of breast cancer lines T47D and MCF-7. We observed that T47D and MCF-7 exhibited a partial EMT phenotype in the presence of activated THP-1 CM. Additionally, MCF-7 displayed a significant increase in migratory and invasive properties. We conclude that M1 secretory factors can promote a partial EMT of epithelial-like breast cancer cells. The targeting of M1 macrophages or their secretory components may inhibit EMT and limit the invasive potential of breast cancer.

Abstract P5-03-06: Novel targets of breast cancer associated with inflammatory tumor microenvironment

Breast cancer affects one in eight women in the USA. Considerable progress in the identification of genetic lesions and their modulation has resulted in newer therapies making breast cancer a manageable disease. However, triple negative breast cancer is still difficult to treat and warrants a search for newer targets. To this end, we focused our attention towards the modulation of the breast cancer epithelium by other cell types such as the endothelial cells and the macrophages. The migratory macrophages and the estrogen sensitive migratory endothelial progenitor cells (EPCs) constitute the cellular milieu within the tumor microenvironment which continuously modulates breast cancer epithelium. We analyzed the interactions of the breast cancer cell lines (MCF-7 and MDA-MB-231) with the highly proliferative human umbilical cord derived CD133+/CD34+/VEGFR-2+ EPCs and M1 polarized macrophages (activated THP-1 cell line) in two separate in vitro studies. The readouts were cell proliferation, changes in epithelial to mesenchymal transition (EMT), and cellular differentiation. We observed morphological and cellular growth changes in the EPCs on treatment with conditioned medium (CM) generated from breast cancer cells, consistent with vasculogenesis and in vitro tubulogenesis. Both, MDA-MB-231 and MCF-7 CM, treatments resulted in enhanced EPCs proliferation and differentiation. However, the differentiation patterns were distinct, with MCF-7 CM increasing the number of cell clusters, whereas MDA-MB-231 CM increasing the number of adherent spindle shaped cells. The paracrine interaction was also assessed with M1 polarized macrophages. We observed decreased cell viability in MCF-7 and MDA-MB-231 cells following activated THP-1 CM and exosome treatments. Analysis of exosomes from activated THP-1 indicated an upregulation of 13 miRNAs compared to unactivated THP-1. The miRNA hsa-miR-146a-5p had the highest upregulation (44 fold increase). This specific miRNA has been observed in senescent cell and it inhibits cell proliferation, suggesting a possible mechanism for exosome-associated growth inhibition. The analysis of the paracrine interactive mediators between breast cancer cells, EPCs, and M1 polarized macrophages is likely to yield viable novel clinically translatable therapeutic targets. Citation Format: Tiwari RK, Ben Rahoma G, Tuli N, Bednarczyk R, Maniyar RR, Chakraborty S, Singh S, Mittelman A, Geliebter J. Novel targets of breast cancer associated with inflammatory tumor microenvironment [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-03-06.

Hyperactive ERK and persistent mTOR signaling characterize vemurafenib resistance in papillary thyroid cancer cells.

Clinical studies evaluating targeted BRAFV600E inhibitors in advanced thyroid cancer patients are currently underway. Vemurafenib (BRAFV600E inhibitor) monotherapy has shown promising results thus far, although development of resistance is a clinical challenge. The objective of this study was to characterize development of resistance to BRAFV600E inhibition and to identify targets for effective combination therapy. We created a line of BCPAP papillary thyroid cancer cells resistant to vemurafenib by treating with increasing concentrations of the drug. The resistant BCPAP line was characterized and compared to its sensitive counterpart with respect to signaling molecules thought to be directly related to resistance. Expression and phosphorylation of several critical proteins were analyzed by Western blotting and dimerization was evaluated by immunoprecipitation. Resistance to vemurafenib in BCPAP appeared to be mediated by constitutive overexpression of phospho-ERK and by resistance to inhibition of both phospho-mTOR and phospho-S6 ribosomal protein after vemurafenib treatment. Expression of potential alternative signaling molecule, CRAF, was not increased in the resistant line, although formation of CRAF dimers appeared increased. Expression of membrane receptors HER2 and HER3 was greatly amplified in the resistant cancer cells. Papillary thyroid cancer cells were capable of overcoming targeted BRAFV600E inhibition by rewiring of cell signal pathways in response to prolonged vemurafenib therapy. Our study suggests that in vitro culture of cancer cells may be useful in assessing molecular resistance pathways. Potential therapies in advanced thyroid cancer patients may combine vemurafenib with inhibitors of CRAF, HER2/HER3, ERK, and/or mTOR to delay or abort development of resistance.

Abstract 3263: Dietary supplement 3, 3’-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer

Tumor angiogenesis refers to the sprouting and cooption of proliferating endothelial cells (EC’s) from adjacent pre-existing host vasculature, and is a key target of cancer therapy. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to promote and support angiogenesis. Within this complex tumor microenvironment, we and others have shown that tumors can recruit bone marrow derived endothelial progenitor cells that differentiate into mature bone marrow-derived endothelial cells and incorporate into sprouting tumor neovessels. Under pathological circumstances, such as breast cancer, a clear association between estrogen receptor expression by EC’s, angiogenic activity, and/or tumor invasiveness has been made. Approximately, 80% of breast cancers are hormone-receptor-positive cancers, thus enabling tamoxifen as the mainstay of breast cancer therapy. The roles of the anti-estrogens fulvestrant (ICI) and the dietary supplement 3, 3’-diindolylmethane (DIM) on cell-cell interaction and angiogenesis have not been fully elucidated. This study is designed to evaluate and compare the effect of these antiestrogens on angiogenesis at the cellular and molecular levels using tube formation of human umbilical vein endothelial cells (HUVEC) as an in vitro angiogenesis model. HUVEC cells were treated with serial dilutions of either DIM or ICI in presence and absence of (3nM) estrogen, and subjected to in vitro tube formation, proliferation, migration, and angiogenesis antibody array assays. We report that HUVEC cells are more sensitive to DIM than ICI. At 25 μM concentration, DIM significantly inhibited the crucial steps of angiogenesis including HUVEC cells proliferation, migration, cytokine release, and tube formation in an estrogen independent manner. On the other hand, at 1 μM concentration, ICI significantly exerted an antiangiogenic effects inhibiting HUVEC cells proliferation, migration, and tube formation, but this effect was totally dependent on the presence of estrogen. These results are validated by our observation that HUVEC cells express estrogen receptor beta (ER-β) and not estrogen receptor alpha (ER-α). A correlative effect between the antiangiogenic activity of DIM and ER-β upregulation was noted. We believe that the anti-estrogenic activity of DIM is mediated through the genomic and non-genomic activity of ER-β in endothelial cells predicting a new target for DIM to manifest its antiangiogenic effect. Citation Format: Ghada M. Ben Rahoma, Neha Y. Tuli, Robert B. Bednarczyk, Rachana R. Maniyar, Abraham Mittelman, Jan Geliebter, Raj Tiwari. Dietary supplement 3, 3’-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3263.

Abstract 4089: Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory cells

Thyroid cancer is the most prevalent endocrine malignancy in the United States with an unacceptably high recurrence rate of 20-30%. Tumor associated macrophages (TAMs), one of the most critical component of solid tumor microenvironments, promote cancer initiation, growth, progression, metastasis and angiogenesis. These TAMs release cytokines as well as other secretory components like exosomes in the tumor microenvironment. Previously, we found that M1 polarized TAMs modulate thyroid cancer phenotype by inducing epithelial-mesenchymal transition (EMT), facilitating tissue metastasis and dissemination. In our present study, we used an in vitro model system to assess the crosstalk between the secretory components of macrophages and the epithelial cells in the thyroid tumor microenvironment. We used THP-1 monocyte/macrophage cell line along with thyroid cancer cell lines: consisting of two papillary cancer cell lines (BCPAP and TPC-1), one anaplastic cancer cell line (8505C) and one follicular cancer cell line (CGTHW-1). We observed that activated THP-1 macrophages are polarized towards M1 phenotype, secreting pro-inflammatory cytokines such as TGF-β, IL6, TNF-α, IL-1β, amongst others. These cytokines are responsible for halt in proliferation and change in morphology to mesenchymal phenotype promoting EMT in thyroid cancer cells. Similar pattern in phenotypical changes were noted in thyroid cancer cells treated with activated THP-1 macrophage exosomes. We also observed that EMT markers, such as vimentin and NFκ-B, are modulated in response to activated macrophage secreted exosomes. Moreover, secretory components from anaplastic thyroid cancer cells led to enhanced activation of THP-1 cells. These findings support a mutual cooperation between thyroid cancer cells and inflammatory cells in tumor microenvironment in defining thyroid cancer phenotype. Such correlation can identify early markers and prevent thyroid cancer differentiation, and are putative targets for therapy. Citation Format: Neha Yashpal Tuli, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari. Thyroid tumor microenvironment: mutual interaction between cancer and inflammatory cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4089.

Abstract 5105: Exosomes secreted by proinflammatory macrophages modulate breast cancer

Multiple cell types reside within the tumor microenvironment (TME) including cancer stem cells, stromal cells, and inflammatory cells. Cellular crosstalk is initiated between these cells through a variety of secreted biomolecules such as cytokines, growth factors, and exosomes. The role of inflammation and numerous secretory factors within the TME have been implicated in the development and progression of various cancers including breast. In this study, we wanted to evaluate the effect of macrophages (a major constituent of inflammation) on breast cancer development. Our study focused on macrophage secreted exosomes which are membraneous nanovesicles that contain various miRNA, mRNA, and proteins. We hypothesized that macrophage secreted exosomes will modulate breast cancer phenotype. An in vitro cell culture system was used in this study to examine macrophage exosome treatments on breast cancer cell lines MCF-7 (ER+) and MDA-MB 231 (ER-, PR-, HER2/neu-). Human monocytic THP-1 monocytes were activated with a common phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) and conditioned media (CM) was collected following 48 h. Exosomes were isolated from THP-1 CM via the use of an exosome isolation reagent and standard centrifugation. Breast cancer cells were then treated with THP-1 exosomes and cell morphology, proliferation, MEK/ERK and PI3K/AKT signaling pathways, and cell cycle were evaluated. MCF-7 and MDA-MB 231 exhibited round and spindle-like morphologies as well as a significant decrease in proliferation at 24 h treatments. Furthermore, MDA-MB 231 displayed increased p-ERK, while MCF-7 showed increased p-MEK following Western blot analysis. Both cell lines exhibited decreased p-AKT and p-S6 kinase expression. Cell cycle analysis showed that MCF-7 and MDA-MB 231 arrested at G2/M at 24 h exosome treatments. THP-1 exosome treatments led to a significant increase in MDA-MB 231 senescence as determined by a senescence assay looking at senescence-associated beta-galactosidase. Increased p21 and decreased Cdc2/Cyclin B1 was also observed which indicates a G2/M arrest. We conclude that specific macrophage secreted exosomes may induce breast cancer cell senescence and affect breast cancer progression. Citation Format: Robert Bronislaw Bednarczyk, Neha Y. Tuli, Ghada Ben Rahoma, Rachana Maniyar, Abraham Mittelman, Raj K. Tiwari. Exosomes secreted by proinflammatory macrophages modulate breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5105.