Jelani C. Zarif

The Androgen Receptor Induces Integrin α6β1 to Promote Prostate Tumor Cell Survival via NF-κB and Bcl-xL Independently of PI3K Signaling

Recent studies indicate that androgen receptor (AR) signaling is critical for prostate cancer cell survival, even in castration-resistant disease wherein AR continues to function independently of exogenous androgens. Integrin-mediated adhesion to the extracellular matrix is also important for prostate cell survival. AR-positive prostate cancer cells express primarily integrin α6β1 and adhere to a laminin-rich matrix. In this study, we show that active nuclear-localized AR protects prostate cancer cells from death induced by phosphoinositide 3-kinase (PI3K) inhibition when cells adhere to laminin. Resistance to PI3K inhibition is mediated directly by an AR-dependent increase in integrin α6β1 mRNA transcription and protein expression. Subsequent signaling by integrin α6β1 in AR-expressing cells increased NF-κB activation and Bcl-xL expression. Blocking AR, integrin α6, NF-κB, or Bcl-xL concurrent with inhibition of PI3K was sufficient and necessary to trigger death of laminin-adherent AR-expressing cells. Taken together, these results define a novel integrin-dependent survival pathway in prostate cancer cells that is regulated by AR, independent of and parallel to the PI3K pathway. Our findings suggest that combined targeting of both the AR/α6β1 and PI3K pathways may effectively trigger prostate cancer cell death, enhancing the potential therapeutic value of PI3K inhibitors being evaluated in this setting.

The importance of non-nuclear AR signaling in prostate cancer progression and therapeutic resistance.

The androgen receptor (AR) remains the major oncogenic driver of prostate cancer, as evidenced by the efficacy of androgen deprivation therapy (ADT) in naïve patients, and the continued effectiveness of second generation ADTs in castration resistant disease. However, current ADTs are limited to interfering with AR ligand binding, either through suppression of androgen production or the use of competitive antagonists. Recent studies demonstrate 1) the expression of constitutively active AR splice variants that no longer depend on androgen, and 2) the ability of AR to signal in the cytoplasm independently of its transcriptional activity (non-genomic); thus highlighting the need to consider other ways to target AR. Herein, we review canonical AR signaling, but focus on AR non-genomic signaling, some of its downstream targets and how these effectors contribute to prostate cancer cell behavior. The goals of this review are to 1) re-highlight the continued importance of AR in prostate cancer as the primary driver, 2) discuss the limitations in continuing to use ligand binding as the sole targeting mechanism, 3) discuss the implications of AR non-genomic signaling in cancer progression and therapeutic resistance, and 4) address the need to consider non-genomic AR signaling mechanisms and pathways as a viable targeting strategy in combination with current therapies.

A phased strategy to differentiate human CD14+monocytes into classically and alternatively activated macrophages and dendritic cells

There are currently several in vitro strategies to differentiate human CD14(+) monocytes isolated from peripheral blood mononuclear cells (PBMCs) into the M1 or M2 macrophage cell types. Each cell type is then verified using flow cytometric analysis of cell-surface markers. Human CD14(+) monocytes have the potential to differentiate into M1 and M2 macrophages, both of which demonstrate varying degrees of cell-surface antigen overlap. Using multiple surface markers with current macrophage polarization protocols, our data reveal several limitations of currently used methods, such as highly ambiguous cell types that possess cell-surface marker overlap and functional similarities. Utilizing interleukin-6 (IL-6) and two phases of cytokine exposure, we have developed a protocol to differentiate human monocytes into M1, M2, or dendritic cells (DCs) with greater efficiency and fidelity relative to macrophages and DCs that are produced by commonly used methods. This is achieved via alterations in cytokine composition, dosing, and incubation times, as well as improvements in verification methodology. Our method reliably reproduces human in vitro monocyte-derived DCs and macrophage models that will aid in better defining and understanding innate and adaptive immunity, as well as pathologic states.

The identification of Macrophage enriched glycoproteins using glycoproteomics

Prostate cancer is a leading cause of cancer-related deaths of men in the United States. Whereas the localized disease is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Immune cells that primarily scavenge debris and promote prostate cancer angiogenesis and wound repair are M2 macrophages. They are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) and have been reported to associate with solid tumors and aide in proliferation, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. To identify novel surface glycoproteins expressed on M2 macrophages, we developed a novel method of creating homogeneous populations of human macrophages from human CD14+ monocytes in vitro. These homogeneous M1 macrophages secrete pro-inflammatory cytokines, and our M2 macrophages secrete anti-inflammatory cytokines as well as vascular endothelial growth factor (VEGF). To identify enriched surface glycoproteins, we then performed solid-phase extraction of N-linked glycopeptides followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on our homogeneous macrophage populations. We discovered five novel peptides that are enriched exclusively on human M2 macrophages relative to human M1 macrophages and human CD14+ monocytes. Finally, we determined whether these surface glycoproteins, found enriched on M2 macrophages, were also expressed in human metastatic castrate-resistant prostate cancer (mCRPC) tissues. Using mCRPC tissues from rapid autopsies, we were able to determine M2 macrophage infiltration by using immunohistochemistry and flow cytometry. These findings highlight the presence of macrophage infiltration in human mCRPC but also surface glycoproteins that could be used for prognosis of localized disease and for targeting strategies.

Abstract 3237: Assessment and targeting of M2-tumor associated macrophages (M2-TAMs) in prostate cancer

Prostate cancer is a leading cause of cancer-related deaths of men in the U.S., and in the past year, over 30,000 men died from this disease. While localized prostate cancer is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Alternatively activated macrophages, also known as M2-macrophages, primarily scavenge debris and in the process, promote angiogenesis and wound repair. M2-macrophages are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors such as prostate cancer to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. The purpose of this project is to develop novel therapeutics that will directly target M2-TAMs for destruction and subsequently attenuate prostate tumor growth, progression and metastasis. The central hypothesis of this study is to determine if targeting of M2-TAMs using specific surface antigens will be an effective therapy for lethal prostate cancer and potentially, other cancers. Our study elucidated M2-Macrophage biology including creating homogenous populations of both M1 and M2 macrophages using a new strategy as to allow for identification of novel surface antigen markers. While using our new method for polarization of human monocyte polarization into macrophages, we found that M2 macrophages polarization was stymied in vitro after glutamine deprivation. We then assessed novel markers of macrophage populations, cytokine and chemokine production, T-Cell inactivation and we also assessed whether these surface antigens that are expressed on M2-macrophages are were found in metastatic human prostate cancers indicative of myeloid infiltration. Validation studies using metastatic prostate cancer tissues from rapid autopsies and surgical specimens supplied by the Department of Urology at Johns Hopkins University School of Medicine were also be performed. Complementary studies will be executed to target these markers for anti-tumor cell efficacy using these relevant model systems. By identifying and targeting specific markers on M2-TAMs, we predict that this targeting will provide a better prognosis for patients who have been diagnosed with lethal prostate cancer. Citation Format: Jelani C. Zarif, James R. Henandez, Kenneth J. Pienta. Assessment and targeting of M2-tumor associated macrophages (M2-TAMs) in prostate 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 3237.

Abstract 2365: Targeting M2-tumor associated macrophages (M2-TAMs) in prostate cancer

Prostate cancer is a leading cause of cancer-related deaths of men in the U.S., and in the past year, over 30,000 men died from this disease. While localized prostate cancer is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Alternatively activated macrophages, also known as M2-macrophages, primarily scavenge debris and in the process, promote angiogenesis and wound repair. M2-macrophages are phenotypically similar to M2-tumor associated macrophages (M2-TAMs) have been reported to associate with solid tumors such as prostate cancer to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. The purpose of this project is to develop novel therapeutics that will directly target M2-TAMs for destruction and subsequently attenuate prostate tumor growth and progression. The central hypothesis of this study is to determine if targeting of M2-TAMs using specific surface antigens will be an effective therapy for lethal prostate cancer and potentially, other cancers. Our study will first elucidate M2-TAM biology, including both a study of known M2-TAM cell surface antigen markers expressed in human prostate cancers as well as rigorous tests to target these markers for anti-tumor cell efficacy using relevant model systems. Functional experiments will then be done in vitro using primary antibodies that are bound by a saporin-conjugated secondary antibody and also, in vivo by utilizing human xenograft mouse models. We will also assess internalization of armed antibodies, M2-TAM cell death, M2-TAM functionality, and EMT status of PCa tumors after targeting. This will be followed by target validation using tissues from rapid autopsies and surgical specimens supplied by the Department of Urology at Johns Hopkins University School of Medicine. By targeting specific markers on M2-TAMs, we predict that this targeting will provide a better prognosis for patients who have been diagnosed with lethal prostate cancer. Modification of targeted ligands and drug combinations will produce a flexible platform for cancer therapy. The studies described here represent an underdeveloped frontier in cancer therapeutics and outline a method of altering the tumor immune microenvironment. If successful, antibodies used against specific M2-TAM surface markers will be constructed into antibody drug conjugates (ADCs). Citation Format: Jelani C. Zarif, James R. Hernandez, Kris F. Sachsenmeier, Robert E. Hollingsworth, Kenneth J. Pienta. Targeting M2-tumor associated macrophages (M2-TAMs) in prostate 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 2365. doi:10.1158/1538-7445.AM2015-2365

Characterization of the macrophage infiltrate in a case of xanthogranulomatous pyelonephritis

Xanthogranulomatous pyelonephritis (XGP) is a rare chronic infectious process of the kidney that results in diffuse destruction of the renal parenchyma [1,2]. The majority of cases of XGP occur in the setting of obstructing nephrolithiasis and are histologically characterized by the presence of lipid-laden foamy macrophages. To date, little is known regarding the molecular and functional phenotype of the infiltrating macrophages observed in cases of XGP. Human CD14+ monocytes can differentiate into macrophages and then become polarized towards two distinct phenotypes [3–5]. Classically activated (also known as M1) macrophages have the ability to secrete pro-inflammatory cytokines such as IFN-γ, IL-2, and TNF-α. Conversely, alternatively activated (also known as M2) macrophages secrete anti-inflammatory cytokines such as IL-10, IL-23, and VEGF. Characterization of the immune infiltrate in cases of XGP is necessary for understanding the pathogenesis of this disease process. Moreover, this knowledge may form the basis of future non-invasive treatment strategies for XGP. Herein, we report the phenotypic characterization of the macrophage infiltrate in a case of XGP.

Abstract B06: Targeting M2-Tumor Associated Macrophages (M2-TAMs) in prostate cancer

Prostate cancer is a leading cause of cancer-related deaths of men in the U.S., and in the past year, over 30,000 men died from this disease. While localized prostate cancer is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Alternatively activated macrophages, also known as M2-macrophages, primarily scavenge debris and in the process, promote angiogenesis and wound repair. M2-macrophages are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors such as prostate cancer to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. The purpose of this project is to develop novel therapeutics that will directly target M2-TAMs for destruction and subsequently attenuate prostate tumor growth, progression, and metastasis. Our hypothesis is to determine if targeting of M2-TAMs by using enriched surface antigens that are targeted by antibody-drug-conjugates (ADCs), be an effective therapy for lethal prostate cancer while simultaneously eliciting an immune response. To identify novel surface antigens expressed on M2-macrophages, we developed a novel method of creating homogenous populations of human macrophages from CD14+ monocytes in vitro. Our homogenous M1 macrophages secrete pro-inflammatory cytokines and our M2 macrophages secrete anti-inflammatory cytokines as well as VEGF. We then performed solid-phase extraction of N-linked glycopeptides (SPEG) followed by liquid chromatography-tandem Mass Spectrometry (LC-MS/MS) on our homogenous macrophage populations. We discovered six novel peptides that are enriched exclusively on M2-macrophages relative to M1 macrophages and CD14+ monocytes. Lastly, we determined if these surface antigens, found enriched on M2 macrophages, were also expressed in human metastatic castrate-resistant prostate cancer (mCRPC). Using mCRPC tissues from rapid autopsies supplied by the Departments of Urology and Surgical Pathology, we were able to determine M2-macrophage infiltration by using immunohistochemistry and flow cytometry. The studies described here outline a method of altering the tumor immune microenvironment. By identifying specific markers on M2-TAMs, we predict that this method of targeting will provide a better prognosis for patients who have been diagnosed with lethal prostate cancer. Citation Format: Jelani C. Zarif, James R. Hernandez, Weiming Yang, Hui Zhang, Kenneth J. Pienta. Targeting M2-Tumor Associated Macrophages (M2-TAMs) in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B06.

Abstract 3704: Targeting M2-tumor associated macrophages (M2-TAMs) using enriched glycoproteins in lethal prostate cancer

Prostate cancer is a leading cause of cancer-related deaths of men in the U.S., and in the past year, over 30,000 men died from this disease. While localized prostate cancer is highly treatable by surgical resection and radiation, cancer that has metastasized remains incurable. Alternatively activated macrophages, also known as M2-macrophages, primarily scavenge debris and in the process, promote angiogenesis and wound repair. M2-macrophages are phenotypically similar to M2 tumor-associated macrophages (M2-TAMs) have been reported to associate with solid tumors such as prostate cancer to facilitate epithelial to mesenchymal transition (EMT), tumor invasiveness, metastasis, and resistance to therapy. As an invasive species within the tumor microenvironment, this makes M2-TAMs an ideal therapeutic target in prostate cancer. The purpose of this project is to develop novel therapeutics that will directly target M2-TAMs for destruction and subsequently attenuate prostate tumor growth, progression, and metastasis. Our hypothesis is to determine if targeting of M2-TAMs by using enriched surface antigens that are targeted by antibody-drug-conjugates (ADCs), be an effective therapy for lethal prostate cancer while simultaneously eliciting an immune response. To identify novel surface antigens expressed on M2-macrophages, we developed a novel method of creating homogenous populations of human macrophages from CD14+ monocytes in vitro. Our homogenous M1 macrophages secrete pro-inflammatory cytokines and our M2 macrophages secrete anti-inflammatory cytokines as well as VEGF. We then performed solid-phase extraction of N-linked glycopeptides (SPEG) followed by liquid chromatography-tandem Mass Spectrometry (LC-MS/MS) on our homogenous macrophage populations. We discovered five novel peptides that are enriched exclusively on M2-macrophages relative to M1 macrophages and CD14+ monocytes. Lastly, we determined if these surface antigens, found enriched on M2 macrophages, were also expressed in human metastatic castrate-resistant prostate cancer (mCRPC). Using mCRPC tissues from rapid autopsies supplied by the Departments of Urology and Surgical Pathology, we were able to determine M2-macrophage infiltration by using immunohistochemistry and flow cytometry. The studies described here outline a method of altering the tumor immune microenvironment. To target M2 macrophages, we used small peptides as well as antibody drug conjugates (ADCs) that targeted the enriched surface glycoproteins expressed on M2-macrophage. We then tested their efficacies in vitro and in a syngeneic prostate tumor mouse model to assess tumor shrinkage and effector and cytotoxic T Cell infiltration. By identifying specific markers on M2-TAMs, we predict that this method of targeting will provide a better prognosis for patients who have been diagnosed with lethal prostate cancer. Citation Format: Jelani C. Zarif, James R. Henandez, Weiming Yang, Hui Zhang, Kenneth J. Pienta. Targeting M2-tumor associated macrophages (M2-TAMs) using enriched glycoproteins in lethal prostate 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 3704. doi:10.1158/1538-7445.AM2017-3704

Abstract 4005: HDAC inhibitors regulate M2 tumor-associated macrophage function through histone acetylation

Macrophage tumor infiltration in metastatic prostate cancer is a predictor of patient prognosis. Macrophages influence tumors in contrasting ways depending on their polarization: M1 macrophages have anti-tumor functions while M2 macrophages are pro-tumor. The external stimuli influencing macrophage differentiation and M2 polarization have been largely elucidated but the resulting downstream changes remain unknown. There are a number of epigenetic differences between M1 and M2 macrophages that act as important functional determinants. Recent work studying these epigenetic changes has implicated histone deacetylases (HDACs), as critical regulators in macrophage differentiation and in maintaining M1 or M2 function. Pan-HDAC inhibitors (HDIs) such as the clinically utilized suberanilohydroxamic acid (SAHA) target a wide range of HDACs and provide a means for manipulating macrophage histone acetylation. Though HDIs have been found to attenuate inflammatory functions in M1 macrophages, the field has yet to explore effects of histone acetylation in M2 macrophages. Using M2 macrophages derived from human CD14 + monocytes, we found that SAHA regulates M2 macrophage polarization and function through alteration of histone acetylation. Work is ongoing to test expression levels of canonical M2 markers such as the mannose receptor CD206 and scavenger receptor CD163 in M2 macrophages exposed to SAHA either during or after M2 polarization. Additionally, investigation into the ability of these populations to secrete M2 cytokines such as IL-10 and VEG-FA and induce EMT in prostate cancer cells is also being pursued. These results contribute to scientific knowledge of epigenetic regulation in macrophage function and elucidate strategies to decrease the pro-tumor function of tumor-infiltrating M2 macrophages. This work lays the foundation for using epigenetic regulators to modulate the tumor microenvironment and advance cancer medicine. Citation Format: Amber E. de Groot, Jelani C. Zarif, Kenneth J. Pienta. HDAC inhibitors regulate M2 tumor-associated macrophage function through histone acetylation [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 4005. doi:10.1158/1538-7445.AM2017-4005