Eunmi Lee

Mimetics of suppressor of cytokine signaling 3: Novel potential therapeutics in triple breast cancer: Mimetics of SOCS3

Suppressor of cytokine signaling (SOCS) family of proteins plays critical role in the regulation of immune responses controlling JAK/STAT mediated inflammatory cytokines. Among the members, SOCS1 and SOCS3 contain a kinase inhibitory region (KIR) and SOCS3 binds to JAK/STAT/gp130 complex by inhibiting the downstream signaling and suppressing inflammatory cytokines. Loss or reduced levels of SOCS3 have been linked to cancer‐associated inflammation and suppressive immunity leading to enhanced tumor growth and metastasis. In line with these reports, we previously demonstrated that proteolytic degradation of SOCS3 in triple negative breast cancer (TNBC) subtype drives the expression of inflammatory cytokines. Therefore, we postulated that SOCS3 mimetics might suppress the inflammatory cytokine production in TNBC subtype and inhibit tumor growth and metastasis. Here we designed and characterized five linear peptides derived from the N‐terminal region of SOCS3 encompassing regions that interface with the JAK2/gp130 complex using the Circular Dichroism and Surface Plasmon Resonance spectroscopies. The KIRESS peptide resulted the sequence containing the most part of the hot‐spots required for binding to JAK2 and was further investigated in vivo in mouse xenografts of MDA‐MB‐231‐luci tumors as models of human TNBC subtype. Expectedly, this peptide showed a significant inhibition of primary tumor growth and pulmonary metastasis. Our studies suggest that SOCS3 peptidomimetics may possess a therapeutic potential in aggressive cancers, such as TNBC subtype, with activated inflammatory cytokines.

Abstract 466: Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model

Metastatic breast cancer is the second leading cause of cancer-related death among women in the United States. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5. MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice. We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naive animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice. Citation Format: Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Abdeljabar El Andaloussi, Ena Novakovic, Raziye Piranlioglu, Mehmet F. Demirci, Shawn G. Clouthier, Max S. Wicha. Systemic early immune priming via tumor-secreted cytokines facilitates breast cancer metastasis in syngeneic mouse model. [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 466. doi:10.1158/1538-7445.AM2015-466

The pleiotropic effects of TNFα in breast cancer subtypes is regulated by TNFAIP3/A20

TNFα is a pleiotropic cytokine which fuels tumor cell growth, invasion, and metastasis in some malignancies, while in others it induces cytotoxic cell death. However, the molecular mechanism by which TNFα exerts its diverse effects on breast cancer subtypes remains elusive. Using in vitro assays and mouse xenografts, we show here that TNFα contributes to the aggressive properties of triple negative breast cancer (TNBC) cell lines via upregulation of TNFAIP3(A20). In a striking contrast, TNFα induces a potent cytotoxic cell death in luminal (ER+) breast cancer cell lines which fail to upregulate A20 expression. Overexpression of A20 not only protects luminal breast cancer cell lines from TNFα-induced cell death via inducing HSP70-mediated anti-apoptotic pathway but also promotes a robust EMT/CSC phenotype by activating the pStat3-mediated inflammatory signaling. Furthermore, A20 overexpression in luminal breast cancer cells induces aggressive metastatic properties in mouse xenografts via generating a permissive inflammatory microenvironment constituted by granulocytic-MDSCs. Collectively, our results reveal a mechanism by which A20 mediates pleiotropic effects of TNFα playing role in aggressive behaviors of TNBC subtype while its deficiency results in TNFα-induced apoptotic cell death in luminal breast cancer subtype.

Abstract 2956: Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation

We previously demonstrated that MDSC subsets accumulated in primary tumor and distant organs regulate tumor plasticity. The mouse transcriptome analysis of in vitro co-cultures and samples from syngeneic mouse model revealed that granulocytic subset of myeloid-derived suppressor cells (gMDSCs) from metastatic 4T1 tumor bearing mice regulate several hundred genes in tumor cells upon co-culture. The top genes are S100A8, S100A9, MMP8, FPR1, CCL3, and TGFβ2 which also predicted poor survival in human solid tumors including breast cancer. Therefore, we called these 6 genes as “metastatic gene signature”. It has been reported that S100A8/S100A9 heterotetramer, called calprotectin, play a key role in inflammation-associated cancer progression. To investigate the role of calprotectin (S100A8/A9) in tumor-mediated immunosuppression and metastasis, we first utilized Tasquinimod, a small molecule targeting S100A9 only. However, Tasquinimod treatment of 4T1 tumor-bearing mice had a moderate anti-tumor activity which may be due to a limited activity on granulocytic MDSC accumulation. In addition, we determined that there was a significant upregulation of S100A8 in MDSCs from Tasquinimod treated mice. This data suggested that inhibiting only S100A9 leads to activation of S100A8 and thus may be ineffective targeting of MDSCs. We therefore run a computational screen of the NCI chemical library against the crystal structure of calprotectin (S100A8/S100A9) and identified 40 lead compounds. We then performed in vitro screening assay to identify compounds that inhibit MDSC induction. We identified 3 compounds that significantly suppressed gMDSC differentiation. We are currently performing in vivo studies with these 3 candidate drugs in our murine breast cancer model and will present our findings at the AACR meeting. We believe that this study will provide a novel drug targeting S100A8/S100A9 heterotetramer, a key molecule in MDSC induction and its regulatory functions during tumor progression. Citation Format: Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El Andaloussi, Sena Arbag, Gang Zhou, Hasan Korkaya. Chemical library screen identifies compounds that target S100A8/S100A9 complex and MDSC accumulation [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 2956. doi:10.1158/1538-7445.AM2017-2956

Abstract 908: Immune regulation of tumor dormancy in syngeneic mouse model

Metastatic disease -end stage of tumor progression- is the major cause of cancer-related death.It is widely accepted that malignant cell plasticity between epithelial-mesenchymal-transition (EMT) and mesenchymal-epithelial-transition (MET) is required for metastasis to occur. The classical model of metastasis suggests tumor cell dissemination occur late in tumor development, however emerging studies strongly indicates that dissemination is an early process and provide a striking evidence that tumor cells start to disseminate during the initial steps of tumor development. Late appearing metastases arise from these early-disseminated tumor cells. The mechanism by which some early-disseminated tumor cells colonize and generate metastatic growth while some remain dormant is not well known. In order to understand the underlying factors, we utilized murine mammary tumors (4T1 as metastatic and EMT6 as less metastatic) in a syngeneic mouse model. We performed time course experiments to determine the early factors that may contribute to the metastatic growth. 4T1 or EMT6 tumor cells were implanted orthotopically into the fat pads and tumor cell dissemination was analyzed over 3 weeks time points. We determined that both 4T1 and EMT tumor cells disseminated as early as one week post-implantation, however only 4T1 tumor cells develop metastasis in distant organs. Furthermore, we also resected primary tumors 1,2 and 3 week post implantation of EMT6-Luci or 4T1-Luci tumors and observed distant metastasis via optical imaging of luciferase expression in live animals. Although the majority of 4T1 tumor bearing mice (>80%) develop pulmonary metastasis when 4T1 tumors resected 2 and 3 weeks post-implantation, only 10% of mice develop metastasis when primary tumor resected one week post implantation. In contrast, EMT6 tumors following resection only relapsed in the primary tumor site but failed to develop metastasis. We investigated the possible mechanism of efficient pulmonary metastatic growth following the resection of tumors 2-3 week post implantation. Interestingly we found a significant infiltration of granulocytic subset of myeloid derived suppressor cells (g-MDSC) in 4T1 tumor bearing mice by week 2 and 3. Furthermore, we found that lung infiltrated g-MDSCs promote tumor cell growth via paracrine factors. In co-culture studies we found that there is a reciprocal secretion of panel of inflammatory cytokines, growth factors and matrix metalloproteases between tumor cell and g-MDSCs suggesting that these cells in the lung microenvironment support the metastatic growth. Our studies provide a new paradigm in the understanding of metastatic growth and the role of microenvironment in distant organs. Citation Format: Raziye Piranlioglu, Maria Ouzounova, Eunmi Lee, Alicia Hudson, Sumeyye Korkaya, Ali Arbab, Hasan Korkaya. Immune regulation of tumor dormancy in syngeneic mouse model. [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 908.

Abstract 1555: MDSC mediated spatiotemporal tumor plasticity in breast cancer metastasis

Metastatic breast cancer is the second leading cause of cancer-related death among women. Although the genetic and epigenetic differences between the metastatic versus non-metastatic breast tumors have been well studied, early events between tumor and immune system in metastatic process remain poorly understood. In order to determine early events, we utilized murine mammary tumors (4T1 as metastatic, EMT6 or 67NR as non-metastatic) in syngeneic mouse model. The 4T1 tumor contained a higher proportion of cancer stem cell (CSC) population compared to the non-metastatic EMT6 or 67NR clones. Although, both murine tumor cell lines (50K each) grow to same size tumors within 8 weeks, 4T1 tumors develop spontaneous metastasis in 100% of animals most of which do not survive more than 8 weeks due to extensive wide spread metastasis to lung, liver and bone. We observed immune infiltrates in the lungs of 4T1 tumor bearing mice as early as 1 week. We next assessed the cytokine profile of metastatic 4T1 tumor compared to non-metastatic counterparts (EMT6 or 67NR) secretes significantly higher levels of inflammatory cytokines, including the IL6, IL8, RANTES, GCSF, GM-CSF, IL12, CXCL16 and CXCL5. MDSCs are potent suppressor of anti-tumor immunity and a significant impediment to cancer therapy. We therefore hypothesized that the tumor secreted inflammatory cytokines promotes the systemic expansion of MDSCs that down regulate immune surveillance and anti-tumor immunity, thus facilitating tumor progression. We sought to determine whether 4T1 tumors could induce MDSCs in mice. Murine 4T1 or EMT6 tumor cells (at 50K cells each) were implanted into the fat pads of BALB/c mice, then sacrificed (4 mice from each group) at weeks 1, 2, 3 and 4 for subsequent evaluation of the MDSC expansion in bone marrow, spleen, lung and tumors. The MDSC induction and infiltration in bone marrow, spleen, lung and tumors were observed as early as one-week post-implantation of 4T1 tumor compared to the EMT6. Furthermore, the MDSCs isolated from 4T1 tumor bearing animals were more suppressive than that of the EMT6 tumor bearing mice. We determined that non-metastatic EMT6-Luciferase tumor growth and metastasis is robustly enhanced in pre-primed animals (in which metastatic 4T1 cells were pre-implanted in the fat pads and resected after 10 days when tumors were 2mm in size) or by IP injection of inflammatory cytokine rich 4T1 conditioned medium when compared to injection of EMT6-Luci cells into naive animals. Our preliminary findings suggested that 4T1 tumors within 10 days of implantation created a systemic tumor-promoting microenvironment and thus promoted the metastatic spread of EMT6-Luci. Together these studies strongly suggest that metastatic 4T1 tumor with high CSC phenotype generate a permissive systemic microenvironment for successful metastasis via secretion of inflammatory cytokines in syngeneic BALB/c mice. Citation Format: Hasan Korkaya, Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El-Andaloussi, Ena Novakovic, Alicia Hudson, Sumeyye Korkaya, Mhmet F. Demirci, Gang Zhou. MDSC mediated spatiotemporal tumor plasticity in breast cancer metastasis. [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 1555.

Abstract 717: Monocytic and granulocytic MDSCs display distinct molecular properties and coordinate the dynamic switches between EMT-MET in breast cancer model

It is widely accepted that the epithelial-mesenchymal plasticity of malignant cells is required during cancer metastatic cascade. The complex phenotypic changes highly depend on the collaboration of various molecular signaling and extracellular cues originating from wide range of stromal cells in the tumor microenvironment. However, the specific mechanisms of how EMT plasticity spatiotemporally regulates metastasis are poorly defined. Myeloid-derived suppressor cells (MDSCs) have been identified in most cancer patients and animal models due to their immune suppressive functions, but recent studies implicate their direct role in promoting metastasis by activating tumor-angiogenesis. To determine the roles of MDSCs in breast cancer metastasis, we utilized murine breast cancer cells, non-metastatic EMT6 and metastatic 4T1 cells. We showed that the metastatic 4T1 murine breast tumors induced early systemic expansion and mobilization of MDSCs in distant sites as well as in the primary tumor. We investigated the direct functions of MDSCs in tumor progression by isolating monocytic and granulocytic MDSCs from primary tumor, lung and bone marrow of tumor-bearing mice and then they were co-cultured with non-metastatic EMT6 cells. We found that tumor infiltrating m-MDSCs from 4T1 tumor-bearing mice increased the expression of Vimentin, Twist1, TGF-â and IL-6 in EMT6 tumor cells. In contrast, flow cytometry sorted lung infiltrating MDSCs from 4T1 tumor-bearing mice enhanced the EpCAM expression and proliferation in EMT6 cells. Cell invasion assay showed that invasive ability of EMT6 cells were significantly increased when they were co-cultured with m-MDSCs while g-MDSCs slightly decreased the number of invaded cells, compared to control group. We utilized immunofluorescence staining and confirmed the increased expression of Vimentin, CK14 (cytokeratin 14) in EMT6 cells co-cultured with m-MDSCs. In contrast, g-MDSCs induced down-regulation of these markers while they increased cell proliferation as assessed by Ki67 staining. Furthermore, flow cytometry analysis showed the increased CD24+CD29+ population, a marker of murine cancer stem cell (CSC) phenotype, in EMT6 cells when co-cultured with m-MDSCs from 4T1 tumor-bearing mice. Tumor sphere assay confirmed that m-MDSCs enhanced sphere forming ability of tumor cells. Taken together, these data suggest that m-MDSCs derived from metastatic 4T1 tumor-bearing mice are able to confer EMT/CSC phenotype on tumor cells while g-MDSCs are more potent in inducing epithelial phenotype and proliferation in tumor cells. Citation Format: Eunmi Lee, Maria Ouzounova, Raziye Piranlioglu, Abdeljabar El Andaloussi, Mehmet Demirci, Ena Novakovic, Alicia Hudson, Sumeyye Korkaya, Gang Zhou, Hasan Korkaya. Monocytic and granulocytic MDSCs display distinct molecular properties and coordinate the dynamic switches between EMT-MET in breast cancer model. [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 717.

Abstract 1554: Myeloid derived suppressor cells-mediated inflammation in metastasis and cancer cachexia

Despite recent advances and better diagnostics, the major challenge is that metastatic breast cancer is still incurable and remains leading cause of cancer related death. Cachexia is considered to be a chronic inflammatory syndrome which is defined by loss of skeletal muscle mass (with or without loss of adipose mass), negative energy and metabolic balance, and systemic inflammation. Cancer patients who develop cachexia are more susceptible to infections and sepsis. Clinical studies suggest that cachexia in cancer patients cannot be fully reversed by conventional nutritional supports, which distinguishes this condition from anorexia. Due to its complexity and lack of clinical biomarkers, currently there is no standard treatment for these patients. Therefore cachexia remains a largely underestimated and untreated condition. Nearly 60-80% of the advanced/ metastatic cancer patients experience cachexia, a condition that accounts for 20% of cancer-related deaths. Chronic inflammation has been recognized as a risk factor contributing to the etiology of many human malignancies. Accumulating evidence suggest that tumor infiltrating immune cells (mainly myeloid origin) differentiate into cells that promote tumor growth and metastasis via inducing a systemic inflammation. Our preliminary studies suggest that systemic induction and infiltration (tumor, bone marrow, spleen, liver, and lung) of myeloid derived suppressor cells (MDSC) generate a pro-inflammatory micro-environment and are a major source of inflammatory cytokines, many of which are implicated in cancer cachexia. Using a murine breast cancer in a syngeneic (immunocompetent) mouse model we show that metastatic (4T1) murine tumor produce significantly higher level of inflammatory cytokines and is able to induce systemic expansion and infiltration of MDSC compared to non-metastatic murine tumor (EMT6). Furthermore, injection of condition media from metastatic 4T1 tumor cells is also able to induce MDSC expansion in vivo suggesting that tumor-produced factors play role in this process. Moreover, we demonstrate the involvement of the inflammatory cytokines in muscle wasting as shown by co-culture experiments with C2C12 myoepithelial cells and analysed the expression of cachexia markers such as E3 ubiquitin ligases Trim63 and Fbxo3, Myh1(myosin heavy chain), Stat3 and NFkB pathway activation, and elevation of pro-cachexia cytokines. Our preliminary studies demonstrated that the monocytic MDSC induce EMT phenotype and contribute to the dissemination of tumor cells while the granulocytic MDSC promote the metastatic outgrowth, and present higher infiltration in the lungs. We therefore propose that tumor-induced inflammatory cytokines play role in induction of MDSC and further elevation of inflammatory markers leading to metastasis and cancer cachexia. Citation Format: Maria Ouzounova, EunMi Lee, Raziye Piranlioglu, Ena Novakovic, Mehmet Demirci, Sumeyye Korkaya, Alicia Hudson, Hasan Korkaya. Myeloid derived suppressor cells-mediated inflammation in metastasis and cancer cachexia. [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 1554.

Abstract 1984: SOCS3 regulates IDO proteasomal degradation and inflammatory signaling in triple negative breast cancer

An intrinsic claudin-low subtype within the TNBC was recently identified based on low/absent expression of luminal differentiation markers, enrichment of epithelial-to-mesenchymal transition (EMT) and stem cell markers. Interleukin-6 (IL6), a potent pleiotropic cytokine, is induced in response to acute and chronic inflammation. IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the Suppressor of cytokine signaling 3 (SOCS3) mediated Stat3/NF-κB pathway. SOCS3 is a critical negative regulator of IL6-mediated pathways. We have showed that constitutive activation of inflammatory loop in transformed cells but not in their untransformed counterparts depends on proteolytic degradation of SOCS3. Several studies indicate that activation of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase (IDO) represents a key pathway suppressing anti-tumor immunity. Moreover a recent study in dendritic cells found that SOCS3 binds IDO via phosphotyrosine-containing peptides and target the IDO/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. IDO is shown to be constitutively expressed by many tumors. We analyzed a panel of breast cancer cell lines and found an inverse correlation between SOCS3 and IDO1 protein levels. Interestingly IDO protein and transcript levels were significantly increased in TNBC compared to luminal cell lines, thus confirming that IL6-JAK-STAT inflammatory loop is responsible for IDO regulation. Enforced expression of stable form of SOCS3, lacking the PEST and SOCS box domain in TNBC cell lines resulted in decreased expression of IDO. Therapeutic targeting of the IDO-IL6-SOCS3 inflammatory loop would revert the immune suppression mediated by IDO in this breast cancer subtype. These studies would provide a strong rationale for development of inflammatory pathway targeted agents for the treatment of TNBC, an aggressive disease that currently lacks molecularly targeted therapeutics. Citation Format: Maria Ouzounova, EunMi Lee, Ena Novakovic, Satish Kumar Noonepalle, Raziye Piranlioglu, Huidong Shi, Max Wicha, Hasan Korkaya. SOCS3 regulates IDO proteasomal degradation and inflammatory signaling in triple negative breast cancer. [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 1984. doi:10.1158/1538-7445.AM2015-1984