Ghada Ben Rahoma

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 714: A case for combining immunotherapy and targeted small molecule inhibitors: Immunoregulation by primary melanoma cells

Metastatic melanoma, leads to the highest number of skin cancer related deaths. Checkpoint inhibitor therapy has witnessed a high success rate in melanoma patients with anti-CTLA-4 and anti-PD-1. However, checkpoint inhibitor molecules and their compensatory stimulatory counterparts are widely expressed on T cells and antigen presenting cells suggesting a robust redundancy in these molecules as clinical targets. Some of these molecules that include CTLA-4, PD-1, HVEM, VISTA, 41-BB, OX-40 and CD226 are also expressed on tumor cells. Their role in regulating an immune response whether it is cell killing or immune evasion remains to be elucidated. We isolated and characterized five primary patient derived melanoma cell lines: MEL-2, MEL-V, 3MM, KFM and GLM2. We screened these cells for the expression of a comprehensive panel of twenty-five co-stimulatory and co-inhibitory molecules by RT-PCR which revealed significant heterogeneity in expression of these molecules compared to normal adult melanocytes under normal conditions; underscoring the importance of understanding tumor tissue pleiotropy prior to designing a therapeutic regimen. Surprisingly, inhibitory molecules including PD-1, VISTA and LAIR1 and stimulatory molecules including 4-1BB, HVEM and ICOS were upregulated differentially in these cell lines by metabolic stress brought on by starvation conditions. Some of these molecules were restored to basal levels of expression on treatment with 10μM vemurafenib (PLX4032), a BRAFV600E inhibitor, for 24 hours. However the treatment led to concurrent upregulation of molecules such as LAG3, BTLA, CD226 and TIM1, suggesting a compensatory mechanism that could aid melanoma adaptation and escape from immune recognition. Exposing melanoma cells to classical activated dendritic cell cytokines, IL-6 and IL-12, led to a differential expression of these molecules. Additionally, experiments using tumor lysate loaded dendritic cells to study activation revealed an ability to modulate immune activation correlating with unique stimulatory and inhibitory molecule expression profile of each primary cell line. Our results underscore the importance of understanding the profile of co-stimulatory and co-inhibitory molecules expressed in tumor cells. With eighty percent of melanoma patients being positive for the BRAFV600E lesion, we make a case for designing a combinatorial therapeutic regimen, with targeted immunotherapies as well as targeting specific genetic lesions with small molecule inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Y. Tuli, Ghada Ben Rahoma, Sarnath Singh, Marc Wallack, Jan Geliebter, Raj K. Tiwari. A case for combining immunotherapy and targeted small molecule inhibitors: Immunoregulation by primary melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 714.

Abstract 5676: Functional pairing of immunomodulatory targets in anaplastic thyroid cancer

Thyroid cancer is the most common type of endocrine malignancy that has an escalating global frequency. Although most well differentiated thyroid cancers (WDTC) are manageable and respond to current therapeutic modalities, undifferentiated anaplastic thyroid cancers (ATC) exhibit a dramatically different clinical behavior and poor prognosis. With the recent development of immunotherapies, targeted, well-defined treatment plans can demonstrate promising treatment outcomes in ATC patients. Precise immunological targets in ATCs with potential clinical relevance are unknown. Major progress has been made in last 5 years toward development of immune checkpoint inhibitors using anti-CTLA-4 and anti-PD-1/PD-L1 antibodies for cancer treatment which has made immunotherapies one of the mainstream treatment choices. Few additional members of the immunoglobulin superfamily of receptors, like LAG3, TIM3 and VISTA have recently been identified as potential checkpoint targets. Interestingly some of these molecules including TIM3 and PD-L1 promote tumor progression and immune escape. Identification of specific immunotherapeutic targets requirs a better understanding of the immune microenvironment in ATC. To this end we evaluated the expression of prominent co-stimulatory and co-inhibitory cell surface molecules by RT-PCR in three thyroid cancer cell lines - TPC-1 (papillary), CGTH-W-1 (follicular) and 8505C (anaplastic). We observed that many co-inhibitory molecules were upregulated in all three tumor cell lines. CTLA4, interestingly, had the highest expression in 8505C. Additionally we observed differential expression of BTLA, LAIR1, TIM3 and VISTA between TPC-1 and 8505C. LAG3, PD-1 and PD-L1 were also upregulated in 8505C compared to TPC-1. Similar pattern was observed with the expression of co-stimulatory molecules, CD40L and GITR . GITR has been shown to have a tumor suppressor function in multiple myeloma. Another co-stimulatory molecule OX40, which has shown promise in tumor recession when targeted, was upregulated in all three cell lines and 8505C showed the highest expression. Our findings suggest that the aggressive and less immunogenic phenotype of ATC might be attributed to the differential expression of these molecules. Targeting these immunomodulatory molecules in ATC warrants a better understanding of the crosstalk between them and it might provide an efficient means for the disease management. Citation Format: Sanjukta Chakraborty, Rachana R. Maniyar, Neha Y. Tuli, Ghada Ben Rahoma, Cameron Budenz, Sarnath Singh, Jan Geliebter, Raj Tiwari. Functional pairing of immunomodulatory targets in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5676. doi:10.1158/1538-7445.AM2017-5676

Abstract 3656: Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells

Melanoma is one of the deadliest forms of skin cancer, with a dim prognosis when metastasized, leading to the highest number of skin cancer related deaths. In recent years there has been a focus on the use of checkpoint inhibitor therapies like anti-CTLA-4, anti-PD-1 and anti-PD-L1 to treat melanoma. PD-L1, HVEM and VISTA expression on cancer cells has been shown to promote immune evasion and tumor survival. CTLA-4, when engaged on tumor cells leads to their apoptosis and LIGHT signaling leads to recruitment of T cells and effective tumor clearance. Thus, since these molecules do not function in isolation, we need to consider tissue pleiotropy and the expression of co-stimulatory and co-inhibitory molecules on tumor tissue. Our laboratory has characterized and established five primary patient derived melanoma cell lines, MEL-2, MEL-V, 3MM, KFM and GLM-2. In an effort to understand tumor tissue pleiotropy, we conducted a comprehensive expression pattern screening of eight co-inhibitory and ten co-stimulatory molecules by RT-PCR. Among others, VISTA, a CD4+ T cell suppressor, HVEM, an immune evasion regulator, LAG3 and TIM3, potential immune checkpoint targets, were seen to be differentially expressed in these primary cell lines. Current therapies target CTLA-4, PD-1 and PD-L1, which also exhibit varying expression in our primary cell lines, pointing to the importance of considering tissue expression of these molecules when administering these novel immunotherapies. Additionally, 80% of melanoma patients are positive for the BRAFV600E genetic lesion and are administered vemurafenib (PLX4032), an inhibitor of the overactive mutated BRAF. Our in vitro studies show that treatment with PLX4032, changed the expression of CTLA-4 and PD-L1 in these primary cells. We observed a decrease in PD-L1 protein expression in MEL-V and GLM-2 and an increase in PD-L1 protein expression in MEL-2 and KFM, the four BRAFV600E positive cell lines. CTLA-4 protein expression demonstrated an upward trend on treatment with PLX4032. This synergy observed between treatment with drugs targeting genetic lesions and the expression of immunomodulatory molecules warrants characterization of tumor biopsies prior to designing an effective combinatorial therapy regime. Our long term goal is to optimize combinatorial immune and drug therapies directed against both co-stimulators and checkpoint inhibitors. Citation Format: Rachana R. Maniyar, Sanjukta Chakraborty, Neha Tuli, Ghada Ben Rahoma, Sarnath Singh, Jan Geliebter, Marc Wallack, Raj K. Tiwari. Implication for checkpoint therapeutics: Expression of co-stimulatory and co-inhibitory molecules in melanoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3656. doi:10.1158/1538-7445.AM2017-3656

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.