Elyse K. Hanly

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.

PLX4032 Mediated Melanoma Associated Antigen Potentiation in Patient Derived Primary Melanoma Cells.

Over expression of various immunogenic melanoma associated antigens (MAAs) has been exploited in the development of immunotherapeutic melanoma vaccines. Expression of MAAs such as MART-1 and gp100 is modulated by the MAPK signaling pathway, which is often deregulated in melanoma. The protein BRAF, a member of the MAPK pathway, is mutated in over 60% of melanomas providing an opportunity for the identification and approval by the FDA of a small molecule MAPK signaling inhibitor PLX4032 that functions to inactivate mutant BRAFV600E. To this end, we characterized five patient derived primary melanoma cell lines with respect to treatment with PLX4032. Cells were treated with 5μM PLX4032 and harvested. Western blotting analysis, RT-PCR and in vitro transwell migration and invasion assays were utilized to determine treatment effects. PLX4032 treatment modulated phosphorylation of signaling proteins belonging to the MAPK pathway including BRAF, MEK, and ERK and abrogated cell phenotypic characteristics such as migration and invasion. Most significantly, PLX4032 led to an up regulation of many MAA proteins in three of the four BRAF mutated cell lines, as determined at the protein and RNA level. Interestingly, MAGE-A1 protein and mRNA levels were reduced upon PLX4032 treatment in two of the primary lines. Taken together, our findings suggest that the BRAFV600E inhibitor PLX4032 has therapeutic potential over and above its known target and in combination with specific melanoma targeting vaccine strategies may have further clinical utility.

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.

The role of surgery in anaplastic thyroid cancer: A systematic review ☆

OBJECTIVE To elucidate the role of surgery in the management of anaplastic thyroid cancer. METHODS Ovid MEDLINE, Cochrane Library, and Google Scholar databases were searched for publications from December 2000 to July 2016. Selection criterion was a focus on the management of anaplastic thyroid cancer in adults. Studies addressing only nonsurgical management and review articles were excluded. Data extraction was performed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Kaplan-Meier analysis was performed on a subset of patients. RESULTS 40 publications were included in the study. Approaches to unresectability and interpretations of resection varied widely. For patients undergoing primary surgery, the median survival was 6.6months. The median survival for non-surgical patients was 2.1months. In the subgroup analysis, the median survival time for patients undergoing surgery was significantly longer in Stage IVB (p=0.022) but not IVC disease. Negative margins did not afford a statistically significant survival benefit. CONCLUSION Surgery is a mainstay of treatment for Stage IVA and IVB disease. For Stage IVC cancer, distant metastasis was not a strict criterion against surgical candidacy among surgeons. The extent of resection and the definition of resectability remain controversial. Negative margins did not significantly increase survival.

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.

Endoscopic resection of nasal schwanomma as a diagnostic and therapeutic adjunct in the treatment of a rare disorder: A case report with literature review

Abstract Isolated nasal schwannomas are rare lesions that can be difficult to diagnose due to nonspecific characteristics. We report a unique case of a patient with a polypoid mass abutting the lacrimal sac found to be a nasal schwanomma. A 66-year-old male with a history of previously resected nasal polyposis returned to the office complaining of symptoms suggestive of rhinosinusitis. Endoscopy was significant for a polypoid mass superior to the inferior turbinate. CT imaging revealed a 2 × 1.8 × 1.3 cm lesion in the right nasal cavity with bony erosion overlying the lacrimal sac. Following endoscopic resection, pathologic analysis revealed a nasal schwanomma based on S100 and Ki-67 staining patterns. The patient has been doing well for over 6 months without recurrence. Although rare, nasal schwanomma should be kept in the differential of any nasal mass, and endoscopic resection can serve as a useful tool in the management of this disorder.

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 5087: Precise characterization of macrophage secretory exosomes can lead to novel therapeutic approaches

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Multiple cell types interact within the tumor microenvironment (TME) through crosstalk events that are mediated by many biomolecules including cytokines, mRNA, and miRNA-containing exosomes that can influence cancer progression as well as suppression. Inflammation within the TME is a major determinant in tumor progression. Since tumor associated macrophages (TAMs) are a major cellular factor in tumor inflammation, we hypothesized that crosstalk events initiated by macrophage secreted factors such as cytokines and exosomes will modulate breast cancer phenotype. We evaluated the cytokine profile of a human monocytic cell line, THP-1, following stimulation with a phorbol ester TPA (12-O-Tetradecanoylphorbol-13-acetate) in vitro. Activated THP-1 cells exhibited an M1 macrophage phenotype. In addition, we assessed the morphological and cellular growth changes of luminal (T47D, MCF-7) and basal-like (MDA-MB 231) breast cancer cell lines in vitro following treatment with activated THP-1 conditioned medium (CM). Each cell line displayed mesenchymal-like features and decreased cellular growth following activated THP-1 CM treatment. MCF-7 and MDA-MB 231 displayed increased MEK/ERK signaling, while all three cell lines showed increased p-AKT expression. Furthermore, epithelial-like T47D and MCF-7 cells exhibited an increase in EMT transcription factor expression. Moreover, although T47D and MCF-7 did not show drastic changes in E-cadherin expression via western blot analysis, immunofluorescence examination showed a decrease in E-cadherin following activated THP-1 CM treatments emphasizing a possible partial EMT event. Lastly, activated THP-1 exosomes were isolated and used to treat MDA-MB 231 to determine the effects on breast cancer cell proliferation and senescence. MDA-MB 231 exhibited significantly decreased cellular growth and increased senescence following incubation with activated THP-1 exosomes. The variety of secretory factors within the TME as a result of inflammation may contribute to tumor suppression as well as tumor progression via a secretion of cytokines and exosomes. Citation Format: Robert Bronislaw Bednarczyk, Yuki Kitadai, Neha Y. Tuli, Elyse K. Hanly, Ghada Benrahoma, Abraham Mittelman, Raj K. Tiwari. Precise characterization of macrophage secretory exosomes can lead to novel therapeutic approaches. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5087. doi:10.1158/1538-7445.AM2015-5087

Abstract 3709: Resistance mechanisms to targeted molecular therapy in thyroid cancer

Thyroid cancer is increasingly common in the United States. Although prognosis is generally very good, thyroid cancer has a high recurrence rate and there are limited treatment options available for advanced metastatic disease. Since genetic mutations causing over-activation of the mitogen-activated protein kinase (MAPK) signaling pathway are found in over 70% of papillary thyroid cancers, molecular inhibitors targeting these kinases are promising therapeutics. A multikinase inhibitor, Sorafenib, was recently FDA-approved for treatment of advanced thyroid cancer as it significantly increased progression-free survival time. To build upon this success, improvement in efficacy and reduction in common side effects may be possible through more targeted approaches. The most common specific mutation to target is BRAFV600E which occurs in 50% of papillary thyroid cancers and 25% of anaplastic thyroid cancers. BRAFV600E is correlated with advanced stage, lymph node metastasis, extrathyroidal extension, resistance to traditional therapy, and cancer-related mortality in papillary thyroid cancer. PLX4032 (Vemurafenib) is a specific kinase inhibitor developed to halt BRAFV600E-mediated excessive activation of the MAPK pathway. BRAFV600E-positive malignant melanoma patients treated with PLX4032 often experience astounding tumor regression followed by development of drug resistance, which is thought to be acquired through signaling via alternative molecules, bypassing the need for BRAFV600E kinase activity. We hypothesized that treatment with PLX4032 would inhibit MAPK signaling and promote an anti-proliferative phenotype in thyroid cancer cell lines harboring BRAFV600E, without having these effects on BRAF-wildtype cells. In BRAF-mutated cells, we also predicted that certain signaling molecules could become activated to circumvent the PLX4032-mediated inhibition, as seen in melanoma. Results demonstrate that PLX4032 inhibits proliferation, causes apoptosis, and arrests cell cycle selectively in BRAFV600E-positive thyroid cells. These phenotypic responses corresponded to diminished phosphorylation of central signaling proteins downstream of BRAF, including MEK, ERK, and mTOR. The mutated papillary thyroid cancer cells express both MAP3K8 (COT) and CRAF, kinases capable of growth-promoting signaling independent of BRAFV600E, in the face of PLX4032 treatment over time. These results indicate that targeted inhibition of BRAFV600E creates powerful anti-tumor effects in thyroid cancer cells, although mechanisms of resistance similar to those seen in melanoma patients may also occur. Detailed investigation into cell signaling pathways that contribute to thyroid cancer progression will allow for more effective combinational targeted molecular therapy and limit development of drug resistance to specific inhibitors such as PLX4032. Citation Format: Elyse K. Hanly, Neha Y. Tuli, Robert Suriano, Robert Bednarczyk, Zbigniew Darzynkiewicz, Augustine L. Moscatello, Edward J. Shin, Jan Geliebter, Raj K. Tiwari. Resistance mechanisms to targeted molecular therapy in thyroid cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3709. doi:10.1158/1538-7445.AM2014-3709