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BackgroundProgrammed cell death protein 1 (PD-1) and CTLA4 combination blockade enhances clinical efficacy in melanoma compared with targeting either checkpoint alone; however, clinical response improvement is coupled with increased risk of developing immune-related adverse events (irAE). Delineating the mechanisms of checkpoint blockade-mediated irAE has been hampered by the lack of animal models that replicate these clinical events.MethodsWe have developed a mouse model of checkpoint blockade-mediated enterocolitis via prolonged administration of an Fc-competent anti-CTLA4 antibody.ResultsSustained treatment with Fc-effector, but not Fc-mutant or Fc-null, anti-CTLA4 antagonist for 7 weeks resulted in enterocolitis. Moreover, combining Fc-null or Fc-mutant CTLA4 antagonists with PD-1 blockade results in potent antitumor combination efficacy indicating that Fc-effector function is not required for combination benefit.ConclusionThese data suggest that using CTLA4 antagonists with no Fc-effector function can mitigate gut inflammation associated with anti-CTLA4 antibody therapy yet retain potent antitumor activity in combination with PD-1 blockade.

// Cara C. Schafer 1 , Yong Wang 1 , Kenneth P. Hough 1 , Anandi Sawant 2 , Stefan C. Grant 1 , Victor J. Thannickal 1 , Jaroslaw Zmijewski 1 , Selvarangan Ponnazhagan 2 , Jessy S. Deshane 1 1 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 2 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA Correspondence to: Jessy S. Deshane, email: treena@uab.edu Keywords: indoleamine 2,3-dioxygenase, myeloid-derived suppressor cells, lung cancer, metabolism, combination therapy Received: March 01, 2016 Accepted: September 13, 2016 Published: September 26, 2016 ABSTRACT Indoleamine 2,3-dioxygenase (IDO) has been implicated in immune evasion by tumors. Upregulation of this tryptophan (Trp)-catabolizing enzyme, in tumor cells and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME), leads to Trp depletion that impairs cytotoxic T cell responses and survival; however, exact mechanisms remain incompletely understood. We previously reported that a combination therapy of gemcitabine and a superoxide dismutase mimetic promotes anti-tumor immunity in a mouse model of lung cancer by inhibiting MDSCs, enhancing polyfunctional response of CD8 + memory T cells, and extending survival. Here, we show that combination therapy targets IDO signaling, specifically in MDSCs, tumor cells, and CD8 + T cells infiltrating the TME. Deficiency of IDO caused significant reduction in tumor burden, tumor-infiltrating MDSCs, GM-CSF, MDSC survival and infiltration of programmed death receptor-1 (PD-1)-expressing CD8 + T cells compared to controls. IDO -/- MDSCs downregulated nutrient-sensing AMP-activated protein kinase (AMPK) activity, but IDO -/- CD8 + T cells showed AMPK activation associated with enhanced effector function. Our studies provide proof-of-concept for the efficacy of this combination therapy in inhibiting IDO and T cell exhaustion in a syngeneic model of lung cancer and provide mechanistic insights for IDO-dependent metabolic reprogramming of MDSCs that reduces T cell exhaustion and regulates anti-tumor immunity.

Background A growing body of evidence indicates a positive correlation between expression of human antimicrobial peptide leucin leucin 37 (LL-37) and progression of epithelial cancers, including prostate cancer (PCa). Although the molecular mechanisms for this correlation has not yet been elucidated, the primary function of LL-37 as a chemotactic molecule for innate immune effector cells suggests its possible association in coordinating protumorigenic mechanisms, mediated by tumor-infiltrating immune cells. Methods To investigate protumorigenic role(s) of cathelicidin-related antimicrobial peptide (CRAMP), a murine orthologue of LL-37, the present study compared tumor growth kinetics between mouse PCa cell lines with and without CRAMP expression (TRAMP-C1 and TRAMP-C1CRAMP-sh, respectively) in immunocompetent mice. CRAMP-mediated chemotaxis of different innate immune cell types to the tumor microenvironment (TME) was observed in vivo and confirmed by in vitro chemotaxis assay. The role of CRAMP in differentiation and polarization of immature myeloid progenitors (IMPs) to protumorigenic type 2 macrophages (M2) in TME was determined by adoptive transfer of IMPs into mice bearing CRAMP(+) and CRAMP(−) tumors. To differentiate protumorigenic events mediated by tumor-derived CRAMP from host immune cell-derived CRAMP, tumor challenge study was performed in CRAMP-deficient mice. To identify mechanisms of CRAMP function, macrophage colony stimulating factor (M-CSF) and monocyte chemoattractant protein 1 (MCP-1) gene expression was analyzed by QRT-PCR and STAT3 signaling was determined by immunoblotting. Results Significantly delayed tumor growth was observed in wild-type (WT) mice implanted with TRAMP-C1CRAMP-sh cells compared to mice implanted with TRAMP-C1 cells. CRAMP(+) TME induced increased number of IMP differentiation into protumorigenic M2 macrophages compared to CRAMP(−) TME, indicating tumor-derived CRAMP facilitates differentiation and polarization of IMPs toward M2. Tumor challenge study in CRAMP deficient mice showed comparable tumor growth kinetics with WT mice, suggesting tumor-derived CRAMP plays a crucial role in PCa progression. In vitro study demonstrated that overexpressed M-CSF and MCP-1 in TRAMP-C1 cells through CRAMP-mediated autocrine signaling, involving p65, regulates IMP-to-M2 differentiation/polarization through STAT3 activation. Conclusion Altogether, the present study suggests that overexpressed CRAMP in prostate tumor initially chemoattracts IMPs to TME and mediates differentiation and polarization of early myeloid progenitors into protumorigenic M2 macrophages during PCa progression. Thus, selective downregulation of CRAMP in tumor cells in situ may benefit overcoming immunosuppressive mechanisms in PCa. Prostate © 2016 Wiley Periodicals, Inc.

BACKGROUND—The antimicrobial peptide, LL-37 (leucine-leucine-37), stimulates proliferation, angiogenesis and cellular migration, inhibits apoptosis and is associated with inflammation. Since these functional processes are often exaggerated in cancer, the aim of the present study was to investigate the expression and role of LL-37 in prostate cancer (PCa) and establish its value as a therapeutic target. METHODS—We evaluated the expression of LL-37 and the murine orthologue, Cathelicidin Related Anti-Microbial Peptide (CRAMP) in human and murine prostate tumors, respectively. Compared to normal/benign prostate tissue, both LL-37 and CRAMP were increasingly overexpressed with advancing grades of primary prostate cancer and its metastasis in human tissues and in the Transgenic Adenocarcinoma Mouse Prostate (TRAMP) model, correspondingly. We subsequently knocked down CRAMP in the highly tumorigenic TRAMP-C1 cell line via a RNA interference (RNAi) strategy to examine the importance of CRAMP on cellular proliferation, angiogenesis, invasion, apoptosis, activation of signaling pathways and tumor kinetics. RESULTS—Abrogation of CRAMP expression led to decreased proliferation, invasion, type IV collagenase, and the amount of phosphorylated Erk1/2 and Akt signaling in vitro. These results were paralleled in vivo. Syngenic implantation of TRAMP-C1 cells subjected to CRAMP knockdown resulted in a decreased tumor incidence and size, and the down regulation of protumorigenic mechanisms. CONCLUSIONS—CRAMP knockdown in a murine prostate cancer model analogously demonstrated the tumorigenic contributions of LL-37 in PCa and its potential as a novel therapeutic target for the treatment of PCa and potentially, other cancers over-expressing the peptide.

Anti-PD1 therapies show a remarkable improvement in response over many standard of care regimens, but there is a significant need to further increase patient responses. Recent clinical studies show improved response rate when combining anti-PD1 and anti-CTLA4 therapies. This provides “proof of concept” that the combination of two immunotherapies can enhance efficacy. The goal of our work was to evaluate the anti-tumor efficacy of B and T lymphocyte attenuator (BTLA) antagonism with anti-PD1 therapy in preclinical mouse tumor models. BTLA is an inhibitory co-receptor that modulates T cell function and is a marker of “exhausted” T cells. The inhibitory signal mediated by BTLA is initiated following engagement with herpesvirus entry mediator (HVEM), a ubiquitous receptor that is highly expressed on malignant cells. One of the challenges to evaluating BTLA in mouse models is the presence of strain-specific allelic polymorphisms. These sequence differences occur adjacent to the HVEM binding sites and can interfere with the ability of certain antibodies to recognize BTLA in different strains of mice or block the HVEM-BTLA signaling. C57BL/6-specific and strain agnostic BTLA binding antibodies are commercially available, but none antagonize HVEM signaling through BTLA in BALB/c mice. To more comprehensively evaluate the anti-BTLA/anti-PD1 combination regimen we generated a first-in-class BALB/c strain polymorphism specific BTLA antagonist monoclonal antibody (40E4) and identified clone PJ196 as a C57BL/6 strain polymorphism specific antagonist. We show that 40E4 and PJ196 not only block BTLA:HVEM interaction as measured by cell ELISA, but also reverse HVEM-mediated suppression in primary T cells from the appropriate mouse strain. An effector function reduced mouse IgG1 (D265A) chimeric form of 40E4 was generated for in vivo use. In combination with anti-PD1 (mDX400), 40E4 mIgG1 (D265A) shows improved tumor growth inhibition (TGI) over mDX400 alone in preclinical subcutaneous mouse models of breast cancer (EMT6) and colon cancer (CT26). To show that anti-BTLA enhancement of the anti-tumor response to mDX400 is not unique to BALB/c mice, we tested the combination of a C57BL/6 BTLA strain-polymorphism specific monoclonal antibody (PJ196) with mDX400. Using the subcutaneous MB49 model of bladder cancer, we show that treatment with PJ196 and mDX400 significantly improved TGI over mDX400 alone. This is the first report evaluating a BTLA antagonist monoclonal antibody in preclinical mouse tumor models and the first report to show BTLA antagonism enhances the effect of anti-PD1 therapy. Citation Format: Nathan R. Miselis, Douglas Linn, Clifford Restaino, Toya Baral, Jane Xia, Roanna Ueda, Anandi Sawant, Jeanne Baker, Gopalan Raghunathan, Xinzhong Wang1, Edward Bowman, Selvakumar Sukumar. Antagonism of the co-inhibitory receptor BTLA enhances efficacy of anti-PD-1 treatment in murine syngeneic tumor models [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 577. doi:10.1158/1538-7445.AM2017-577

Chemoresistance due to heterogeneity of the tumor microenvironment (TME) hampers the long-term efficacy of first-line therapies for lung cancer. Current combination therapies for lung cancer provide only modest improvement in survival, implicating necessity for novel approaches that suppress malignant growth and stimulate long-term antitumor immunity. Oxidative stress in the TME promotes immunosuppression by tumor-infiltrating myeloid-derived suppressor cells (MDSC), which inhibit host protective antitumor immunity. Using a murine model of lung cancer, we demonstrate that a combination treatment with gemcitabine and a superoxide dismutase mimetic targets immunosuppressive MDSC in the TME and enhances the quantity and quality of both effector and memory CD8+ T-cell responses. At the effector cell function level, the unique combination therapy targeting MDSC and redox signaling greatly enhanced cytolytic CD8+ T-cell response and further decreased regulatory T cell infiltration. For long-term antitumor effects, this therapy altered the metabolism of memory cells with self-renewing phenotype and provided a preferential advantage for survival of memory subsets with long-term efficacy and persistence. Adoptive transfer of memory cells from this combination therapy prolonged survival of tumor-bearing recipients. Furthermore, the adoptively transferred memory cells responded to tumor rechallenge exerting long-term persistence. This approach offers a new paradigm to inhibit immunosuppression by direct targeting of MDSC function, to generate effector and persistent memory cells for tumor eradication, and to prevent lung cancer relapse. Cancer Res; 73(22); 6609–20. ©2013 AACR.

Immune cells and their secreted growth factors play major roles in tumor growth and metastasis. Interplay between the growing tumor and infiltrating immune cells determines the nature of immune response and ultimately, tumor fate. Increased infiltration of protumorigenic immune cells promotes tumor growth as well as dissemination to distant sites. These cells induce immunosuppression that inhibits proliferation and functions of cells of antitumor immune response. One population of immunosuppressive cells that is increasingly gaining attention is myeloid-derived suppressor cells (MDSC). MDSCs are immature myeloid progenitors that suppress T-cell effector functions and promote angiogenesis. MDSC numbers are elevated at both the primary tumor and metastatic sites, including bone. In addition to immunosuppressive functions of MDSCs, we and others have recently discovered a novel function for MDSCs as osteoclast progenitors. Osteolysis is a common complication in the carcinomas of breast, lung, prostate, and multiple myeloma with poor prognosis. Therefore, targeting the functions of MDSCs may exert dual therapeutic effects on immunosuppression and bone pathology. Cancer Res; 73(15); 4606–10. ©2013 AACR.

Elevated levels of plasmacytoid dendritic cells (pDC) have been observed as breast cancer disseminates to the bone. The selective depletion of pDC in mice led to a total abrogation of bone metastasis as well as to an increase in TH1 antitumor response, suggesting that pDC may be considered as a potential therapeutic target for metastatic breast cancer.

Myeloid-derived suppressor cells (MDSC) from mice bearing bone metastases differentiate into functional osteoclasts in vitro and in vivo, through a signaling pathway that relies on nitric oxide. In addition, MDSC-targeting drugs have been shown to robustly inhibit osteolysis. Thus, MDSC stand out as novel osteoclast progenitors and hence as candidate targets for the control of osteolytic bone disease.

Blockade of the checkpoint inhibitor PD-1 has demonstrated remarkable success in the clinic for the treatment of a growing list of different cancers. However, several tumor types are resistant to anti-PD-1 monotherapy. This observation has spurred numerous combination studies to reveal what additional therapeutic interventions may complement anti-PD1 blockade. Recently it has been shown that immunogenic cell death (ICD), induced by radiation and/or chemotherapy, improves T cell responses against different tumor types. ICD is characterized by damage-associated molecular patterns (DAMPs) including surface expression of calreticulin, and release of ATP and HMGB1. Recognition of DAMPs triggers dendritic cell maturation and functions that are critical for tumor antigen-specific T cell activation. Thus therapies that evoke ICD may further augment anti-tumor immunity elicited by anti-PD-1. In mouse syngeneic tumor models, we observed combinatorial anti-tumor activity of anti-PD1 and the cyclin-dependent kinase inhibitor, dinaciclib. We hypothesized that dinaciclib potentiates the effects of anti-PD-1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib express the hallmarks of ICD including HMGB1 and ATP release and surface expression of calreticulin. Dinaciclib treatment also increases tumor cell phagocytosis and induces dendritic cell maturation. Furthermore, mice immunized with dinaciclib-treated tumor cells are resistant to subsequent tumor challenge. Tumors from mice receiving anti-PD1 and dinaciclib have increased T cell infiltration and dendritic cell activation, indicating the overall quality of the immune response generated may be improved with the combo. Taken together, these findings suggest a potential mechanism for the observed synergy between dinaciclib and anti-PD1. Dinaciclib induces immunogenic cell death, converting the tumor cell into an endogenous vaccine and thereby boosting the effects of anti-PD-1. Citation Format: Dewan Mohammed Sakib Hossain, Fernando Ugarte, Anandi Sawant, Mingmei Cai, Venkataraman Sriram, Elaine Pinheiro, Svetlana Sadekova, Alissa Chackerian. Dinaciclib induces immunogenic cell death and enhances anti-PD-1 mediated tumor suppression. [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 562.

Enhanced bone destruction is a hallmark of various carcinomas such as breast cancer, where osteolytic bone metastasis is associated with increased morbidity and mortality. Immune cells contribute to osteolysis in cancer growth, but the factors contributing to aggressive bone destruction are not well understood. In this study, we show the importance of myeloid-derived suppressor cells (MDSC) in this process at bone metastatic sites. Because MDSC originate from the same myeloid lineage as macrophages, which are osteoclast precursors, we hypothesized that MDSC may undergo osteoclast differentiation and contribute to enhanced bone destruction and tumor growth. Using an immunocompetent mouse model of breast cancer bone metastasis, we confirmed that MDSC isolated from the tumor-bone microenvironment differentiated into functional osteoclasts both in vitro and in vivo. Mechanistic investigations revealed that nitric oxide signaling was critical for differentiation of MDSC into osteoclasts. Remarkably, osteoclast differentiation did not occur in MDSC isolated from control or tumor-bearing mice that lacked bone metastasis, signifying the essential cross-talk between tumor cells and myeloid progenitors in the bone microenvironment as a requirement for osteoclast differentiation of MDSC. Overall, our results identify a wholly new facet to the multifunctionality of MDSC in driving tumor progression, in this case as a novel osteoclast progenitor that specifically drives bone metastasis during cancer progression. Cancer Res; 73(2); 672–82. ©2012 AACR.

Purpose: Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow–derived mesenchymal stem cells have the ability to differentiate into osteoblast and produce osteoprotegerin, a decoy receptor for the receptor activator for nuclear factor κB ligand, naturally. The present study examined the potential of unmodified mesenchymal stem cells to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer. Experimental Design: The human prostate cancer cell line PC3 was implanted in tibiae of severe combined immunodeficient mice. After establishment of the tumor, either unmodified or genetically engineered mesenchymal stem cells overexpressing osteoprotegerin was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro–computed tomography, immunohistochemistry, and histomorphometry. Results: Data indicated significant (P < 0.001) inhibition of tumor growth and restoration of bone in mice treated with unmodified and modified mesenchymal stem cells. Detailed analysis suggested that the donor mesenchymal stem cell inhibited tumor progression by producing woven bone around the growing tumor cells in the tibiae and by preventing osteoclastogenesis. Conclusions: Overcoming the limitation of the number of mesenchymal stem cells available in the bone can provide significant amelioration for osteolytic damage without further modification. (Clin Cancer Res 2009;15(23):7175–85)

Heterogeneity and immunosuppression in the tumor microenvironment (TME) has dampened the efficacy of current frontline combination chemotherapies for lung cancer which has provided limited improvement in patient survival. Modified amino acid metabolism in the TME contributes to lung cancer progression and suppression of anti-tumor immunity. Enhanced tryptophan metabolism in primary lung cancer patients is indicative of advanced disease stage. One of the key regulators of tryptophan metabolism is an intracellular, heme-containing enzyme indoleamine 2,3-dioxygenase (IDO). IDO catabolizes the breakdown of tryptophan into a toxic, immunosuppressive metabolite, L-Kynurenine (L-Kyn). Induction of IDO and accumulation of its metabolite correlates with poor prognosis and overall patient survival. We hypothesized that IDO enzymatic activity would be elevated in lung cancer patients compared to normal healthy controls. We predicted that combination chemotherapy strategies would modulate serum levels of L-Kyn and IDO activity in the immune compartment of lung cancer patients. Using an IDO enzymatic activity assay to measure the production of L-Kyn, we demonstrate that prior to receiving combination chemotherapies, Non-Small Cell Lung Cancer (NSCLC) patients (n=11) display significantly elevated levels of this immunosuppressive serum metabolite compared to healthy relatives (n=8) (Student's t-test p=0.000087; Paired Means p=0.0001). Following the second cycle of treatment, serum L-Kyn was reduced significantly (Student's t-test p=0.023; Paired Means p=0.043) in a subset of patients (n=3 of 5) suggesting response to chemotherapy, but these post-treatment levels remained elevated overall compared to healthy relatives. Additionally, greater levels of IDO enzymatic activity were observed in circulating immunosuppressive myeloid-derived suppressor cells from NSCLC patients and peripheral blood mononuclear cells prior to receiving combination chemotherapy compared to those from healthy relatives. These preliminary studies suggest that the IDO enzymatic pathway and tryptophan metabolism may serve as important therapeutic targets and diagnostic markers in predicting clinical outcomes of combination chemotherapies in the treatment of NSCLC in humans. Citation Format: Cara C. Schafer, Yong Wang, Anandi Sawant, Tong Huan Jin, Selvarangan Ponnazhagan, Stefan C. Grant, Jessy S. Deshane. Combination chemotherapy significantly reduces indoleamine 2,3-dioxygenase activity in NSCLC patients. [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 634. doi:10.1158/1538-7445.AM2014-634

CD8+ T cells are cytolytic effectors of adaptive immunity against invading pathogens, and under various pathological conditions including cancer. In syngeneic, immunocompetent murine models of epithelial cancers, including breast and lung cancer, we observed up-regulation in frequencies of a unique CD8+ T cell population, characterized as CD8+CD25-Foxp3+, referred here as CD8+ natural regulatory T cells. This regulatory cell population was present in very high numbers during initial stages of tumor growth. However, a drastic reduction in CD8+ natural regulatory T cells was observed as tumor burden increased, and also with the onset of metastasis to distant sites, including bone in the case of breast cancer. Upon further investigation of these cells, we observed that they are highly cytotoxic towards the cancer cells. Adoptive transfer of these CD8+ natural regulatory cells obtained from mice with minimal tumor burden resulted in significantly reduced tumor growth in the tumor-bearing recipients. There was also an inverse relationship between CD8+ natural regulatory T cells and plasmacytoid dendritic cells (pDC) as tumor burden increased. Additionally, depletion of pDC, either using depletion antibody or by using a transgenic mouse model lacking pDC, showed increased numbers of CD8+ natural regulatory T cells that resulted in decreased tumor burden. Analysis of a possible molecular mechanism on the role of CD8+ natural regulatory T cells as anti-tumor effectors demonstrated that these cells express PD-1. Furthermore, PD-L1, a ligand for PD-1, is expressed by both pDC and cancer cells in vivo. Ongoing experiments are focused on deciphering a possible role of PD-1 and PD-L1 interaction in determining the fate of CD8+ natural regulatory T cells in the presence of pDC and cancer cells through in vitro assays and using transgenic murine models and depletion antibodies for both PD-1 and PD-L1. Collectively, these data suggest a novel anti-tumor immune population that has a potential to be used as immunotherapy for cancer in conjunction with conventional treatments. Citation Format: Anandi Sawant, Carnella Lee, Cara Schafer, Jessy Deshane, Selvarangan Ponnazhagan. CD8+ natural regulatory T cells as antitumor immune effectors in carcinomas of breast and lung. [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 2794. doi:10.1158/1538-7445.AM2014-2794

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Successful treatments for patients with metastatic PCa are limited suggesting a need for alternate targets. Recently, we identified that antimicrobial peptides in human (LL-37) and mouse (CRAMP) are positively correlated with PCa progression in humans and mice, respectively. As a host defense peptide, LL-37/CRAMP functions against microbial pathogens, and in chemotaxis of leukocytes at sites of inflammation. Preliminary studies pertaining to immunomodulation of PCa cell-derived CRAMP in situ indicated that CRAMP chemoattracts myeloid-derived suppressor cells (MDSC), a heterogeneous population of myeloid precursors that induce T cell suppression, to tumor microenvironment (TME) indicating the significance of CRAMP in pro-tumorigenic immune cell functions during PCa progression. The present study further evaluated role of CRAMP as an immunomodulator during PCa growth using transplantable mouse PCa cell line, TRAMP-C1, containing varying CRAMP expression in wild-type and CRAMP loss of function mouse (Cnlp-/-) models. Results of the study indicated targeted down-regulation of CRAMP in mouse PCa cells delays tumorigenesis in syngenic immunocompetent mouse model suggesting significance of CRAMP in PCa development. Whereas implantation of PCa cells abrogated of CRAMP resulted in retention of neutrophils and macrophages in spleen at significantly elevated levels, mice bearing CRAMP-expressing tumors displayed decreased number of neutrophils and macrophages in spleen, but increased infiltration toward TME. These results suggest tumor-derived CRAMP possibly mediates infiltration of neutrophils and macrophages toward TME from spleen. Whether the infiltrated neutrophils and macrophages correlate to pro-tumorigenic N2 and M2 cells, respectively, needs to be confirmed. Since host immune cells, mainly neutrophils, produce CRAMP and recruit additional innate effectors to inflammatory sites, we further analyzed whether host immune cell-derived CRAMP exert synergistic effects in PCa growth together with tumor-derived CRAMP by using Cnlp-/- mice. Results indicated not only comparable tumor growth kinetics, but also comparable levels of MDSC and neutrophils in TME between Cnlp-/- and wild-type mice, suggesting that tumor is a key source of CRAMP that functions as a chemoattractant of MDSC and neutrophils. Taken together, present study proposes that tumor-produced CRAMP functions as a chmoattractant for infiltration of pro-tumorigenic immune cells; MDSC, neutrophils, and macrophages that facilitate tumor growth. Citation Format: Ha-Ram Cha, Anandi Sawant, Jonathan Hensel, Carnella Lee, Selvarangan Ponnazhagan. Immunomodulatory role of CRAMP (cathelicidin-related antimicrobial peptide) in prostate 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 162. doi:10.1158/1538-7445.AM2014-162

Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis.

We have recently demonstrated that the combination of gemcitabine and a superoxide dismutase mimetic protects mice against lung cancer by suppressing the functions of myeloid-derived suppressor cells and by activating memory CD8+ T-cell responses. Persistent memory cells exhibited a glycolytic metabolism, which may have directly enhanced their effector functions. This combinatorial therapeutic regimen may reduce the propensity of some cancer patients to relapse.

Breast cancer (BCa) is the most common malignancy in United States women, accounting for greater than 40,000 deaths each year. Studies have attributed the presence of cancer stem cells (CSC) with resistance to chemotherapy as a key component of refractories of treatments in BCa. Breast cancer stem cells (BCa-SC) are characterized by CD24lo and CD44hi phenotype with mesenchymal properties, which differ from the epithelial CD24hi and CD44hi phenotype expressed in the majority of BCa cells. Thus, further understanding properties of BCa-SC may lead to the development of novel treatment options towards treating refractory cancer cells. Fundamental to this, further characterization of BCa-SC will be needed to identify therapeutic targets based CSC biology. To this end, we sought to characterize the plasticity of BCa-SC based on cell cycle in vitro and growth kinetics in vivo. A murine BCa cell line, 4T1, was cultured in CSC medium and analyzed every 6 hrs for CD24 and CD44 expression by flow cytometry. Results of this analysis indicated that specific BCa-SC markers peaked every 24 hours. This data supports a possible reprogramming of BCa-SC based on cell cycle regulation, suggesting the use of cell cycle inhibitors to block cells in particular stages of the cell cycle, which may improve therapeutic approaches to induce cell death. The stemness of these cells was further confirmed by real-time PCR. BCa-SC showed increased expression of ALDH1, TWIST, and TGF-β3 with decreased E-Cadherin and CD24 levels correlating to the phenotypic changes observed with CD24 and CD44 expression. To further examine the role of BCa-SC in tumor growth in vivo, BALB/c mice were subcutaneously implanted with populations of 4T1 cells that constituted CSC and non- cancer stem cells (NCSC) in different combinations and tumor growth was analyzed. Results showed that the growth kinetics CSC and NCSC when used alone was significantly lower in vivo compared to mixed populations containing both CSC and NCSC, suggesting CSC may attribute for the repopulation of the disease but an aggressive growth requires differentiated non-stem cells in the tumor microenvironment. Citation Format: Carnella M. Lee, Anandi Sawant, Ha-Ram Cha, Selvarangan Ponnazhagan. Growth characteristics of breast cancer stem cells in vitro and in vivo. [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 250. doi:10.1158/1538-7445.AM2013-250

Prostate cancer (PCa) is the most common type of cancer among males in the United States. However, even with advanced diagnostic capacity and conventional therapies such as surgery and chemotherapy, the 5-year survival rate for disseminated disease is approximately 36 months suggesting the need for finding alterate targets and therapies to increase patient survival. We have recently demonstrated that LL-37, an antimicrobial peptide that plays an important role in innate immune response against microbial pathogens, and its mouse orthologue CRAMP (Cathelicidin-related antimicrobial peptide) plays an important role in the progression of PCa both in humans and in a spontaneously developing mouse PCa model, respectively. Over-expression of CRAMP has also been recently reported in a few human epithelial cancers including carcinomas of the breast and lung. Results of our recent studies also demonstrated that targeted knock-down of CRAMP expression in a mouse PCa cell line, derived from the transgenic adenocarcinoma mouse prostate (TRAMP) model, TRAMP C1, resulted in decreased level of myeloid-derived suppressor cells (MDSC) in the spleen upon syngeneic transplantation. Based on these findings, the present study determined the role of LL-37/CRAMP in immune modulation during PCa progression. C57BL/6 mice were subcutaneously implanted with 3 different PCa cell lines; TRAMP C1 (CRAMPhigh), TRAMP C1scrambled (CRAMPhigh) and TRAMP C1CRAMPshRNA (CRAMPlow). Mice in TRAMP C1 and TRAMP C1scrambled groups developed measurable tumors starting from 45 days post-implantation, whereas mice implanted with TRAMP C1CRAMPshRNA showed measurable tumors from 120 days post-implantation suggesting that high levels of CRAMP expression in TRAMP C1 and TRAMP C1scrambled groups promote prostate tumor growth in vivo. Immune cell phenotyping was performed on cells collected from spleen and tumor at day 50 and 55 post-implantation to identify modulation in infiltrating immune cells as a surrogate for decrease in tumor growth due to abrogation of CRAMP expression. The result indicated a higher number of neutrophils infiltrating to the tumor site from the spleen during the initial stages of tumor growth in TRAMP C1 and TRAMP C1scrambled groups, while high numbers of neutrophils remained in the spleen of TRAMP C1CRAMPshRNA group during that time point. Elevated numbers of MDSC were observed both in spleen and in tumor microenvironment in TRAMP C1 and TRAMP C1scrambled groups suggesting that MDSC may further promote PCa growth whereas mice in the TRAMP C1CRAMPshRNA group showed significantly low level of MDSC in spleen, even when compared to control mice, at earlier time point. The number of plasmacytoid dendritic cells (pDC) both in spleen and tumor in TRAMP C1 and TRAMP C1scrambled groups was low while significantly high numbers of pDC were observed in spleen in TRAMP C1CRAMPshRNA group suggesting that high numbers of pDC may suppress the tumor growth by exerting pro-inflammatory response through cytokines such as IL-12. Altogether, the data suggest that CRAMP promotes PCa growth and plays an important role in immunomodulation during PCa progression. The CRAMP induces infiltration of neutrophils to tumor site and increased number of MDSC in both spleen and tumor, which may induce immunosuppression. Since CRAMP triggers protumorigenic stimuli including angiogenesis, invasion and key immune modulation to promote the tumor growth, LL-37/CRAMP may be used as a possible therapeutic target for PCa treatment. Ongoing tumor challenge studies in CRAMP knockout mouse model will shed more light to confirm these observations and to possibly extend the findings in human patients as a potential therapeutic target. Citation Format: Ha-Ram Cha, Anandi Sawant, Carnella Lee, Jonathan Hensel, George Tsuladze, Selvarangan Ponnazhagan. Immunomodulatory effects of LL-37/CRAMP in prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B86.

Our recent studies indicated a significant up-regulation of anterior gradient protein-2 (AGR-2) in bone metastatic human prostate cancer cells, PC3, following growth in bone marrow conditioned medium. Evidence indicates that AGR2 belongs to the family of protein disulfide isomerases and has been linked to intestinal mucus production, tumor growth and metastasis of various adenocarcinomas and poor survival of patients with breast and prostate cancers. Few recent reports have also reported the regulatory elements of AGR2 function, but its specific role(s) in tumorigenesis and metastasis is still unclear. In the present study, we have analyzed the role of AGR2 in cancer progression and metastasis using prostate cancer as a model. Human prostate cancer tissue microarrays and autopsy tissues, obtained from University of Alabama at Birmingham consortiums were immunostained for AGR-2 expression. Results were also confirmed in a spontaneously developing transgenic adenocarcinoma of mouse prostate (TRAMP) model. Further characterization of AGR-2 function was determined in PC3 cells with targeted silencing of AGR-2 expressions for tumor growth and metastases in vitro and in vivo. Results of our study found AGR-2 expression mainly in the prostate luminar epithelial cells, but no difference was observed between normal prostate gland, BPH and early stages of prostate cancer in both human and TRAMP mouse. Significant reduction in AGR-2 expression was observed in grade IV & V prostate cancers. Interestingly, although declining AGR-2 was evident in higher grade prostate tumors, significantly elevated AGR-2 expressions were observed in the metastatic sites of the same individuals. AGR-2-silenced PC3 cells formed tumors in SCID mice similar to control cells whereas their ability to remain attached to various ECM proteins were greatly compromised. Several key integrins, which mediates ECM binding, were also found to be down-regulated in the AGR-2 silenced PC3 cells, suggesting loss of AGR-2 may be associated with their detachment from the primary tumor and initiation of metastasis. AGR-2 is further up-regulated in metastatic sites indicating its necessity for restoration of integrins for attachment in a new microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5173. doi:1538-7445.AM2012-5173

Prostate cancer has the highest rate of incidence among all cancers in men in the United States and the second leading cause of death behind lung cancer. The human homologue of the frog (Xenopus) anterior gradient protein (AGR-2) has gained considerable attention in recent years for its putative role in neoplastic growth, metastasis and poor survival of patients with breast and prostate cancers. AGR-2 is reported to be androgen dependent and expressed only during the early stages of prostate cancer progression, whereas significant decline in AGR-2 levels in higher grades tumors coincided with metastatic disease. Human primary prostate cancer tissues and regional metastasis were immunostained for AGR-2 expression. Results indicated that AGR-2 was localized in the glandular epithelium of the prostate gland. No significant difference in AGR-2 staining was observed between normal, BPH and well-differentiated prostate cancers, except in normal prostate, where AGR-2 was mostly concentrated in the nuclei. Similar results were observed in a spontaneously developing transgenic adenocarcinoma of mouse prostate (TRAMP) model. AGR-2 expressions was reduced significantly in moderately and poorly-differentiated tumors in both human and TRAMP mice. Higher AGR-2 levels were found in the metastatic tissue albeit lower expressions in the highest grades prostate tumors within the same individual, suggesting further requirement of AGR-2 for growth at the metastatic sites. Targeted silencing of AGR-2 in a bone metastatic prostate cancer cell line, PC3, resulted in significantly reduced cellular adhesion to various extracellular matrix proteins and a failure to form acinar structures following 3-dimensional growth on matrigel. We also determined that AGR-2 silencing led to down-regulation of crucial integrins in PC3 cells, which possibly explains mechanism by which AGR-2 maintains polarized morphology of epithelial cells through promotion of cell adhesion. Based on our findings, we hypothesize that loss of AGR-2 in prostate cancer cells allows detachment from the basement membrane of the primary site and initiates metastasis. AGR-2 as well as integrin levels are restored during passage through circulation and at the metastatic site(s), helping them re-attach to the new microenvironment. Citation Format: Diptiman Chanda, Anandi Sawant, Jonathan Adam Hensel, Tatyana Isayeva, Stephanie D. Reilly, Gene P. Siegal, Claire Smith, Selvarangan Ponnazhagan. Loss of anterior gradient protein in high-grade prostate cancer is associated with loss of tumor cell adhesion and epithelial cell polarity [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B52.

Skeletal metastasis is common in late stage prostate cancer, which leads to significant decline in the quality of life and mortality. In a recent study, we identified over-expression of anterior gradient protein (AGR2) in the PC3, bone metastatic prostate cancer cells following in vitro growth in bone marrow-conditioned medium. AGR2 is a secreted protein and originally reported for its role in the formation of forebrain and mucus-secreting cement gland in the frog Xenopus laevis. Role of AGR2 in carcinogenesis and metastasis is recently being investigated and reports indicate it is over-expressed in various adenocarcinomas and linked to poor survival. But no study has elucidated the role of AGR2 in bone metastasis. When AGR2 expression was compared between human prostate cancer cell lines obtained from bone (PC3), lymph node (LnCap) and brain (Du145) by RT-PCR and Western blot, highest level of AGR2 was observed in PC3 cells. To further confirm, PC3 cells expressing firefly luciferase were injected in athymic nude mice via intra-cardiac route and cells were retrieved from bone, adrenal, lung and heart 3 weeks later following organ dissemination. RT-PCR analysis indicated highest AGR2 expression in PC3 cells isolated from bone microenvironment, suggesting that AGR2 may have a potential role in prostate cancer bone metastasis. To correlate the relevance of this finding in humans, AGR2 immunohistochemistry was performed on human primary and metastatic prostate cancer tissue sections. Preliminary results indicate higher AGR2 expression in metastasized bone sections compared to primary prostate cancers. Additionally, AGR2 was stably silenced in the PC3 cells utilizing a short hairpin RNA and growth kinetics of the cell line was tested in vivo following intra-cardiac injection in a preclinical mouse model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5261. doi:10.1158/1538-7445.AM2011-5261

Prostate cancer commonly metastasizes to the skeleton in advanced stages and leads to significant decline in life expectancy. In a recent study we demonstrated enhanced proliferation of the human prostate cancer cell line PC3 when cultured in the presence of bone marrow conditioned medium (BMCM). In order to identify key molecules involved in increasing proliferation rate, total RNA was isolated from PC3 cells grown in either BMCM or regular medium and subjected to cDNA microarray analysis. Results indicated a significant up-regulation of anterior gradient homolog 2 (AGR2) in the PC3 cells cultured in BMCM compared to those grown in regular medium. AGR2 is a secreted protein and originally reported for its involvement in the formation of forebrain and mucus-secreting cement gland in the frog Xenopus laevis. AGR2 is also highly expressed in human adenocarcinomas of the prostate, breast, esophagus and the pancreas and a study indicated poor survival of prostate cancer patients with higher AGR2 expression. We compared AGR2 gene expression by RT-PCR in different metastatic prostate cancer cell lines, isolated from the bone (PC3 and C4-2B), brain (Du145) and the lymph node (LnCap). Highest AGR2 expression was observed in bone metastatic cell lines, mainly PC3 cells and least expression was observed in Du145 cells. PC3 cells expressing firefly luciferase were also injected via intra-cardiac route into 6 week-old SCID mice and widespread metastases were observed in the lung, heart, adrenal and bone. Three weeks after the inoculation of the PC3 cells, the mice were sacrificed, tumor cells were harvested from various metastatic sites, total RNA isolated and subjected to RT-PCR analysis for AGR2 expression. Interestingly, highest AGR2 expression was observed in PC3 cells isolated from bone microenvironment, suggesting that AGR2 may have a potential role in prostate cancer bone metastasis. To test this hypothesis, we developed a subclone of PC3 cells with targeted knock down of AGR2 transcripts using lentivirus shRNA vector and tested the cell clone in vitro by implanting intra-tibially or subcutaneously in SCID mice. Results of this study indicated a failure of tumor development in AGR2 knocked down PC3 clones. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 231.