Erandi Rajagurubandara

Determinants of Sequence-Specificity within Human AID and APOBEC3G

Human APOBEC3G (A3G) and activation-induced deaminase (AID) belong to a family of DNA-cytosine deaminases. While A3G targets the last C in a run of Cs, AID targets C in the consensus sequence WRC (W is A or T and R is a purine). Guided by the structures of the A3G carboxyl-terminal catalytic domain (A3G-CTD), we identified two potential regions (region 1 and region 2) that may interact with DNA and swapped the corresponding regions between a variant of A3G-CTD and AID. The resulting hybrids were expressed in Escherichia coli and two different genetic assays and a biochemical assay were used to determine the sequence selectivity of the hybrids in promoting C to T mutations. The results show that while the 10 amino acid region 2 of A3G was its principal sequence-specificity determinant, region 1 of A3G enhanced the target cytosine preference conferred by region 2. In contrast, neither of the two regions in AID individually or in combination were sufficient to confer the DNA sequence preference of this protein upon A3G. Instead, introduction of AID sequences in A3G relaxed the sequence-specificity of the latter protein. Our results show that the sequence selectivity of APOBEC family of enzymes is determined by at least two separate sequence segments and there may be additional regions of the protein involved in DNA sequence recognition.

Bone marrow fat: linking adipocyte-induced inflammation with skeletal metastases

Adipocytes are important but underappreciated components of bone marrow microenvironment, and their numbers greatly increase with age, obesity, and associated metabolic pathologies. Age and obesity are also significant risk factors for development of metastatic prostate cancer. Adipocytes are metabolically active cells that secrete adipokines, growth factors, and inflammatory mediators; influence behavior and function of neighboring cells; and have a potential to disturb local milleu and dysregulate normal bone homeostasis. Increased marrow adiposity has been linked to bone marrow inflammation and osteoporosis of the bone, but its effects on growth and progression of prostate tumors that have metastasized to the skeleton are currently not known. This review focuses on fat-bone relationship in a context of normal bone homeostasis and metastatic tumor growth in bone. We discuss effects of marrow fat cells on bone metabolism, hematopoiesis, and inflammation. Special attention is given to CCL2- and COX-2-driven pathways and their potential as therapeutic targets for bone metastatic disease.

Macrophage cathepsin K promotes prostate tumor progression in bone

Bone marrow macrophages (BMMs) share common progenitors with osteoclasts and are critical components of bone–tumor microenvironment; however, their function in prostate tumor growth in the skeleton has not been explored. BMMs are the major source of inflammatory factors and proteases, including cysteine protease cathepsin K (CTSK). In this study, utilizing mice deficient in CTSK, we demonstrate the critical involvement of this potent collagenase in tumor progression in bone. We present the evidence that tumor growth and progression in the bone are impaired in the absence of CTSK. Most importantly, we show for the first time that BMM-supplied CTSK may be involved in CCL2- and COX-2-driven pathways that contribute to tumor progression in bone. Together, our data unravel novel roles for CTSK in macrophage-regulated processes, and provide evidence for close interplay between inflammatory, osteolytic and tumor cell-driven events in the bone–tumor microenvironment.

Marrow adipocyte-derived CXCL1 and CXCL2 contribute to osteolysis in metastatic prostate cancer

Increased bone marrow adiposity is a common feature of advanced age, obesity and associated metabolic pathologies. Augmented numbers of marrow adipocytes positively correlate with dysregulated bone remodeling, also a well-established complication of metastatic disease. We have shown previously that marrow adiposity accelerates prostate tumor progression in the skeleton and promotes extensive destruction of the bone; however, the factors behind adipocyte-driven osteolysis in the skeletal tumor microenvironment are not currently known. In this study, utilizing in vivo diet-induced models of bone marrow adiposity, we reveal evidence for positive correlation between increased marrow fat content, bone degradation by ARCaP(M) and PC3 prostate tumors, and augmented levels of host-derived CXCL1 and CXCL2, ligands of CXCR2 receptor. We show by in vitro osteoclastogenesis assays that media conditioned by bone marrow adipocytes is a significant source of CXCL1 and CXCL2 proteins. We also demonstrate that both the adipocyte-conditioned media and the recombinant CXCL1 and CXCL2 ligands efficiently accelerate osteoclast maturation, a process that can be blocked by neutralizing antibodies to each of the chemokines. We further confirm the contribution of CXCR2 signaling axis to adiposity-driven osteoclastogenesis by blocking fat cell-induced osteoclast differentiation with CXCR2 antagonist or neutralizing antibodies. Together, our results link CXCL1 and CXCL2 chemokines with bone marrow adiposity and implicate CXCR2 signaling in promoting effects of marrow fat on progression of skeletal tumors in bone.

Bone marrow adipocytes promote the Warburg phenotype in metastatic prostate tumors via HIF-1α activation

Metabolic adaptation is increasingly recognized as a key factor in tumor progression, yet its involvement in metastatic bone disease is not understood. Bone is as an adipocyte-rich organ, and a major site of metastasis from prostate cancer. Bone marrow adipocytes are metabolically active cells capable of shaping tumor metabolism via lipolysis and lipid transfer. In this study, using in vitro and in vivo models of marrow adiposity, we demonstrate that marrow fat cells promote Warburg phenotype in metastatic prostate cancer cells. We show increased expression of glycolytic enzymes, increased lactate production, and decreased mitochondrial oxidative phosphorylation in tumor cells exposed to adipocytes that require paracrine signaling between the two cell types. We also reveal that prostate cancer cells are capable of inducing adipocyte lipolysis as a postulated mechanism of sustenance. We provide evidence that adipocytes drive metabolic reprogramming of tumor cells via oxygen-independent mechanism of HIF-1α activation that can be reversed by HIF-1α downregulation. Importantly, we also demonstrate that the observed metabolic signature in tumor cells exposed to adipocytes mimics the expression patterns seen in patients with metastatic disease. Together, our data provide evidence for a functional relationship between marrow adipocytes and tumor cells in bone that has likely implications for tumor growth and survival within the metastatic niche.

Photoactivated inhibition of cathepsin K in a 3D tumor model

Abstract Collagenolytic activity of cathepsin K is important for many physiological and pathological processes including osteoclast-mediated bone degradation, macrophage function and fibroblast-mediated matrix remodeling. Here, we report application of a light-activated inhibitor for controlling activity of cathepsin K in a 3D functional imaging assay. Using prostate carcinoma cell line engineered to overexpress cathepsin K, we demonstrate the utility of the proteolytic assay in living tumor spheroids for the evaluation and quantification of the inhibitor effects on cathepsin K-mediated collagen I degradation. Importantly, we also show that utilizing the ruthenium-caged version of a potent nitrile cathepsin K inhibitor (4), cis-[Ru(bpy)2(4)2](BF4)2 (5), offers significant advantage in terms of effective concentration of the inhibitor and especially its light-activated control in the 3D assay. Our results suggest that light activation provides a suitable, attractive approach for spatial and temporal control of proteolytic activity, which remains a critical, unmet need in treatment of human diseases, especially cancer.

Adipocyte-activated oxidative and ER stress pathways promote tumor survival in bone via upregulation of Heme Oxygenase 1 and Survivin

Metastatic tumor cells engage the local tumor microenvironment and activate specific pro-survival mechanisms to thrive and progress in the harsh bone marrow niche. Here we show that the major contributors to the survival of carcinoma cells that have colonized the bone marrow are the adipocyte-induced oxidative stress and ER stress pathways. We demonstrate that upon exposure to adipocyte-rich environments in vitro or in vivo, bone-trophic prostate and breast tumor cells upregulate the oxidative stress enzyme, HO-1. We also show that HO-1 levels are significantly increased in human metastatic prostate cancer tissues and that stable HO-1 overexpression in tumor cells promotes growth and invasiveness. Co-incident with the adipocyte-induced expression of HO-1, there is an upregulation of ER chaperone BIP and splicing of XBP1, indicating adipocyte-driven unfolded protein response, a process that we show to be sensitive to antioxidant treatment. Importantly, we also demonstrate that triggering of the oxidative stress and ER stress responses, or HO-1 induction by adipocyte exposure result in the activation of pro-survival pathways, involving survivin. Collectively, our findings reveal a new link between HO-1 and survivin expression in tumor cells, and provide a new insight into potentially targetable survival pathways in bone-metastatic disease.

The Lipid Side of Bone Marrow Adipocytes: How Tumor Cells Adapt and Survive in Bone

Purpose of ReviewBone marrow adipocytes have emerged in recent years as key contributors to metastatic progression in bone. In this review, we focus specifically on their role as the suppliers of lipids and discuss pro-survival pathways that are closely linked to lipid metabolism, affected by the adipocyte-tumor cell interactions, and likely impacting the ability of the tumor cell to thrive in bone marrow space and evade therapy.Recent FindingsThe combined in silico, pre-clinical, and clinical evidence shows that in adipocyte-rich tissues such as bone marrow, tumor cells rely on exogenous lipids for regulation of cellular energetics and adaptation to harsh metabolic conditions of the metastatic niche. Adipocyte-supplied lipids have a potential to alter the cell’s metabolic decisions by regulating glycolysis and respiration, fatty acid oxidation, lipid desaturation, and PPAR signaling. The downstream effects of lipid signaling on mitochondrial homeostasis ultimately control life vs. death decisions, providing a mechanism for gaining survival advantage and reduced sensitivity to treatment.SummaryThere is a need for future research directed towards identifying the key metabolic and signaling pathways that regulate tumor dependence on exogenous lipids and consequently drive the pro-survival behavior in the bone marrow niche.

Abstract LB-315: Bone marrow adipocytes alter the metabolic phenotype of metastatic prostate cancer cells through the activation of HIF-1a

Bone is a preferential site of metastasis from prostate cancer (PCa). Age and obesity, conditions that increase adipocyte numbers in bone marrow, are risk factors for skeletal metastases from PCa. Research in our laboratory focuses on understanding the interactions between adipocytes and tumor cells that have infiltrated the bone marrow. Specifically, we are examining how the secretion, transport, and uptake of adipocyte-supplied factors promote metastatic progression in bone. We have previously shown that bone marrow adipocytes enhance a glycolytic phenotype in PCa cells through paracrine activation of glycolytic enzymes GLUT1, HK2, PDK1, ENO2, and LDHa at both the mRNA and protein levels. This correlated with an increase in glycolytic activity as measured by higher levels of lactate secretion and decreases in oxidative phosphorylation, indicative of an enhanced Warburg-like metabolic signature. We then assessed HIF-1a activity through mRNA analysis of HIF-1a target genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF). We found that CA9 and VEGF had elevated expression levels in tumor cells exposed to adipocytes, indicative of enhanced HIF-1a activity. Notably, PCa cells exposed to bone marrow adipocytes had significantly enhanced presence of HIF-1a in the nucleus, result further confirming HIF-1α, activation in tumor cells exposed to marrow adipocytes. Hypoxia chamber experiments revealed that these effects were observable regardless of the presence or absence of oxygen, indicating the presence of oxygen-independent HIF-1a signaling or a state of pseudohypoxia in tumor cells. These results were further validated in multiple in vitro cell culture and in vivo mouse models, where an increased production of both glycolytic genes and HIF-1α target genes was shown to correlate with marrow adiposity. Our overall hypothesis is that bone marrow adipocyte-supplied lipids within the bone microenvironment cause HIF-1a activation and metabolic switch to glycolysis in metastatic PCa cells in bone, leading to their increased aggressiveness and resistance to therapy. Citation Format: Jonathan Diedrich, Erandi Rajagurubandara, Mackenzie Herroon, Izabela Podgorski. Bone marrow adipocytes alter the metabolic phenotype of metastatic prostate cancer cells through the activation of HIF-1a. [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 LB-315.

Abstract A18: Investigating the role of bone marrow adiposity on macrophage function and osteoclast differentiation

Prostate cancer (PCa) is the second leading cause of cancer-related deaths among men. Although PCa is slow to progress, patients eventually develop metastases at distant sites, predominantly bone. Once established in the bone marrow, PCa becomes a devastating and incurable disease. The emerging evidence suggests that overweight and obesity are associated with shorter time to disease recurrence and poor patient survival. One major consequence of obesity is the accumulation of adipocytes within the bone marrow, a process which we have previously shown to accelerate tumor progression in the skeletal sites. We demonstrated that prostate tumor cells exposed to bone marrow adipocyte-derived factors (Adipo CM) exhibit lipid accumulation, increased invasiveness, and changes in cell morphology that promote an invasive phenotype. We have also shown that increased marrow adiposity results in augmented levels of tumor-promoting inflammatory factors in bone. Therefore, in the present study, we investigated the effects of adiposity on bone marrow macrophage (BMM) function, phenotype, and differentiation to mature osteoclasts. Utilizing a co-culture system of BMMs and PCa cells, we demonstrate that BMMs exposed to Adipo CM have increased expression and secretion of osteopontin (OPN), a key regulator of macrophage function that has been implicated in prostate tumor progression. We also observed that BMMs are more invasive toward PCa cells exposed to Adipo CM. Our studies show that IL-10 expression, a chemokine regulated by OPN, is suppressed in BMMs co-cultured with Adipo CM and PCa cells. Conversely, arginase-1 and CD163, markers of M2 macrophage phenotype, are highly induced by macrophage-tumor cell interactions. Our results suggest a direct role for OPN in regulation of BMM phenotype in the bone-tumor microenvironment. Furthermore, our investigations of adiposity-driven bone remodeling in tumor progression in bone reveal that adipocyte-derived factors accelerate BMM differentiation into osteoclasts as evidenced by TRAcP staining, overexpression of osteoclastogenesis-associated genes, and increased proteolytic cleavage of DQ-collagen I. Utilizing functional fluorescence-based assays and western blotting approaches we also show that expression and proteolytic activity of cathepsin K, a bone-degrading cysteine protease, is upregulated in osteoclasts differentiating in the presence of Adipo CM. This further supports the involvement of adipocyte-derived factors in tumor-induced bone disease. In summary, our work highlights novel roles of adipocyte-derived factors in macrophage function, behavior, and osteoclastogenesis in the bone marrow niche. Studies are currently underway to determine how lipid uptake affects their function and tumor-promoting activities in bone. Citation Format: Aimalie Lynnette Hardaway, Mackenzie K. Herroon, Erandi N. Rajagurubandara, Izabela Podgorski. Investigating the role of bone marrow adiposity on macrophage function and osteoclast differentiation. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A18. doi:10.1158/1538-7445.CHTME14-A18