Hawley C. Pruitt

microRNA-29 negatively regulates EMT regulator N-myc interactor in breast cancer

BackgroundN-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis.MethodWeb based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3β phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses.ResultsInvasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/β-catenin signaling resulting from inactivation of GSK3β.ConclusionAberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.

IL-11 produced by breast cancer cells augments osteoclastogenesis by sustaining the pool of osteoclast progenitor cells

BackgroundInterleukin (IL)-11, a cytokine produced by breast cancer, has been implicated in breast cancer-induced osteolysis (bone destruction) but the mechanism(s) of action remain controversial. Some studies show that IL-11 is able to promote osteoclast formation independent of the receptor activator of NF-κB ligand (RANKL), while others demonstrate IL-11 can induce osteoclast formation by inducing osteoblasts to secrete RANKL. This work aims to further investigate the role of IL-11 in metastasis-induced osteolysis by addressing a new hypothesis that IL-11 exerts effects on osteoclast progenitor cells.MethodsTo address the precise role of breast cancer-derived IL-11 in osteoclastogenesis, we determined the effect of breast cancer conditioned media on osteoclast progenitor cells with or without an IL-11 neutralizing antibody. We next investigated whether recombinant IL-11 exerts effects on osteoclast progenitor cells and survival of mature osteoclasts. Finally, we examined the ability of IL-11 to mediate osteoclast formation in tissue culture dishes and on bone slices in the absence of RANKL, with suboptimal levels of RANKL, or from RANKL-pretreated murine bone marrow macrophages (BMMs).ResultsWe found that freshly isolated murine bone marrow cells cultured in the presence of breast cancer conditioned media for 6 days gave rise to a population of cells which were able to form osteoclasts upon treatment with RANKL and M-CSF. Moreover, a neutralizing anti-IL-11 antibody significantly inhibited the ability of breast cancer conditioned media to promote the development and/or survival of osteoclast progenitor cells. Similarly, recombinant IL-11 was able to sustain a population of osteoclast progenitor cells. However, IL-11 was unable to exert any effect on osteoclast survival, induce osteoclastogenesis independent of RANKL, or promote osteoclastogenesis in suboptimal RANKL conditions.ConclusionsOur data indicate that a) IL-11 plays an important role in osteoclastogenesis by stimulating the development and/or survival of osteoclast progenitor cells and b) breast cancer may promote osteolysis in part by increasing the pool of osteoclast progenitor cells via tumor cell-derived IL-11. However, given the heterogeneous nature of the bone marrow cells, the precise mechanism by which IL-11 treatment gives rise to a population of osteoclast progenitor cells warrants further investigation.

Roles of N‐Myc and STAT Interactor in Cancer: From Initiation to Dissemination

N‐myc & STAT Interactor, NMI, is a protein that has mostly been studied for its physical interactions with transcription factors that play critical roles in tumor growth, progression and metastasis. NMI is an inducible protein, thus its intracellular levels and location can vary dramatically, influencing a diverse array of cellular functions in a context‐dependent manner. The physical interactions of NMI with its binding partners have been linked to many aspects of tumor biology including DNA damage response, cell death, epithelial‐to‐mesenchymal transition and stemness. Thus, discovering more details about the function(s) of NMI could reveal key insights into how transcription factors like c‐Myc, STATs and BRCA1 are contextually regulated. Although a normal, physiological function of NMI has not yet been discovered, it has potential roles in pathologies ranging from viral infection to cancer. This review provides a timely perspective of the unfolding roles of NMI with specific focus on cancer progression and metastasis.

Developing Cures: Targeting Ontogenesis in Cancer

Cancer has long been known to histologically resemble developing embryonic tissue. Since this early observation, a mounting body of evidence suggests that cancer mimics or co-opts developmental processes to facilitate tumor initiation and progression. Programs important in both normal ontogenesis and cancer progression broadly fall into three domains: the lineage commitment of pluripotent stem cells, the appropriation of primordial mechanisms of cell motility and invasion, and the influence of multiple aspects of the microenvironment on the parenchyma. In this review we discuss how derangements in these developmental pathways drive cancer progression with a particular focus on how they have emerged as targets of novel treatment strategies.

Methods to Evaluate Cell Growth, Viability, and Response to Treatment in a Tissue Engineered Breast Cancer Model

The use of in vitro, engineered surrogates in the field of cancer research is of interest for studies involving mechanisms of growth and metastasis, and response to therapeutic intervention. While biomimetic surrogates better model human disease, their complex composition and dimensionality make them challenging to evaluate in a real-time manner. This feature has hindered the broad implementation of these models, particularly in drug discovery. Herein, several methods and approaches for the real-time, non-invasive analysis of cell growth and response to treatment in tissue-engineered, three-dimensional models of breast cancer are presented. The tissue-engineered surrogates used to demonstrate these methods consist of breast cancer epithelial cells and fibroblasts within a three dimensional volume of extracellular matrix and are continuously perfused with nutrients via a bioreactor system. Growth of the surrogates over time was measured using optical in vivo (IVIS) imaging. Morphologic changes in specific cell populations were evaluated by multi-photon confocal microscopy. Response of the surrogates to treatment with paclitaxel was measured by optical imaging and by analysis of lactate dehydrogenase and caspase-cleaved cytokeratin 18 in the perfused medium. Each method described can be repeatedly performed during culture, allowing for real-time, longitudinal analysis of cell populations within engineered tumor models.

BH3 mimetics suppress CXCL12 expression in human malignant peripheral nerve sheath tumor cells

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive, Schwann cell-derived neoplasms of the peripheral nervous system that have recently been shown to possess an autocrine CXCL12/CXCR4 signaling loop that promotes tumor cell proliferation and survival. Importantly, the CXCL12/CXCR4 signaling axis is driven by availability of the CXCL12 ligand rather than CXCR4 receptor levels alone. Therefore, pharmacological reduction of CXCL12 expression could be a potential chemotherapeutic target for patients with MPNSTs or other pathologies wherein the CXCL12/CXCR4 signaling axis is active. AT101 is a well-established BCL-2 homology domain 3 (BH3) mimetic that we recently demonstrated functions as an iron chelator and thus acts as a hypoxia mimetic. In this study, we found that AT101 significantly reduces CXCL12 mRNA and secreted protein in established human MPNST cell lines in vitro. This effect was recapitulated by other BH3 mimetics [ABT-737 (ABT), obatoclax (OBX) and sabutoclax (SBX)] but not by desferrioxamine (DFO), an iron chelator and known hypoxia mimetic. These data suggest that CXCL12 reduction is a function of AT101s BH3 mimetic property rather than its iron chelation ability. Additionally, this study investigates a potential mechanism of BH3 mimetic-mediated CXCL12 suppression: liberation of a negative CXCL12 transcriptional regulator, poly (ADP-Ribose) polymerase I (PARP1) from its physical interaction with BCL-2. These data suggest that clinically available BH3 mimetics might prove therapeutically useful at least in part by virtue of their ability to suppress CXCL12 expression.

Conditional knockout of N-Myc and STAT interactor disrupts normal mammary development and enhances metastatic ability of mammary tumors

The process of organ development requires a delicate balance between cellular plasticity and differentiation. This balance is disrupted in cancer initiation and progression. N-Myc and STAT interactor (NMI: human or Nmi: murine) has emerged as a relevant player in the etiology of breast cancer. However, a fundamental understanding of its relevance to normal mammary biology is lacking. To gain insight into its normal function in mammary gland, we generated a mammary-specific Nmi knockout mouse model. We observed that Nmi protein expression is induced in mammary epithelium at the onset of pregnancy, in luminal cells and persists throughout lactation. Nmi knockout results in a precocious alveolar phenotype. These alveoli exhibit an extensive presence of nuclear β-catenin and enhanced Wnt/β-catenin signaling. The Nmi knockout pubertal ductal tree shows enhanced invasion of the mammary fatpad and increased terminal end bud numbers. Tumors from Nmi null mammary epithelium show a significant enrichment of poorly differentiated cells with elevated stem/progenitor markers, active Wnt/β-catenin signaling, highly invasive morphology as well as, increased number of distant metastases. Our study demonstrates that Nmi has a distinct role in the differentiation process of mammary luminal epithelial cell compartment and developmental aberrations resulting from Nmi absence contribute to metastasis and demonstrates that aberration in normal developmental program can lead to metastatic disease, highlighting the contribution and importance of luminal progenitor cells in driving metastatic disease.

Abstract A11: N-Myc and STAT interactor (NMI): A regulator of developmental signaling and EMT

The NMI protein associates with known key transcription factors such as Myc and STATs and serves as a critical regulatory co-factor that dictates specialized functions. While a physiological function for NMI is yet unknown, it has potential roles in pathologies ranging from viral infection to cancer. We will present pioneering work from our laboratory showing that NMI impacts two major developmental signaling pathways - TGFβ and Wnt pathways. EMT is one of the key phenomena underlying normal embryonic and organ development, with a vital role in metastatic progression. We discovered that the expression of NMI is significantly downregulated in metastatic breast tumors. We recapitulated this by stably silencing the expression of NMI in breast cancer cells. Abrogating NMI expression from epithelial-like breast cancer cells enabled cells to assume a mesenchymal-like phenotype. Detailed molecular and functional investigations revealed that this mesenchymal transition was facilitated by decreased STAT5 signaling that caused concomitant reduction in SMAD7 expression and manifestation of a TGFβ-driven EMT program. In contrast, breast cancer cells restored for NMI expression showed autophagic vacuoles and LC3 processing. We found that NMI expression restricts Wnt/β-catenin signaling by upregulation of the secreted Wnt inhibitor, DKK1. Thus NMI prompts activation of GSK3-β, a key kinase upstream of the TSC1/TSC2 complex. Inhibition of GSK3-ββin NMI expressing cells activated mTOR signaling and decreased the cells’ autophagic response. Autophagy is a determinant of cellular survival through dormancy and cytotoxic drug insult. Rapid progression of autophagy leads to cell death. In agreement with our molecular analyses, we determined that abrogation of NMI expression rendered cells resistant to cisplatin and doxorubicin. Our findings elicit interesting possibilities about the role of NMI in regulating EMT and autophagy. Evidence from our investigations strongly suggests that loss of NMI leading to aberrant activation of multiple developmental signaling pathways, may be a vital event in the progression of breast cancer characterized by reduced autophagy and stabilization of transcription factors that function as major drivers of EMT. Citation Format: Rajeev S. Samant, Hawley C. Pruitt, Brandon J. Metge, Lalita A. Shevde. N-Myc and STAT interactor (NMI): A regulator of developmental signaling and EMT. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A11.

Abstract 657: N-Myc and STAT Interactor knock out in the mammary epithelium prompts hyper-proliferation

N-Myc and STAT Interactor (NMI) is an evolutionarily conserved protein that is widely expressed in fetal and adult tissues. Early studies implicated its role in regulating the activities of transcription factors (such as MYC, STATs, BRCA1, TIP60 etc.) critical to tumor progression and metastasis. However, the functional relevance of these regulatory activities of NMI remains unknown. Recent findings from our lab have revealed that NMI protein expression is decreased by 70% in primary tumor specimens from patients with metastatic breast cancer. Most recently, we have found that lack of NMI expression in breast cancer cells confers resistance to chemotherapy by blocking autophagy-induced cell death. Thus the status of NMI expression in breast cancer patients may be an important clinical consideration. Additionally, our functional studies have demonstrated that loss of NMI expression allows manifestation of TGFβ and Wnt driven EMT that results in increased invasion and metastatic dissemination. Overall, we have noticed a profound impact of NMI on multiple developmental signaling pathways that are essential for mammary development as well as tumor progression. To further elucidate the biological role of NMI, we have created a genetically engineered mammary specific Nmi knock out model. We observed that in normal murine mammary tissue Nmi is expressed in the mammary epithelium during all stages of mammary development. However, it9s expression is strikingly induced at the onset of pregnancy, implicating an important role of NMI in mammary ductal development and/or lactation. Remarkably, the Nmi knock out mice exhibit distinctly increased number of alveolar structures (30% more than in control mice) during lactation. Moreover, these Nmi-/- mammary glands also show 20% more proliferating mammary epithelial cells (MECs) compared to respective littermate controls. In addition, 3D-alveologenesis of Nmi knock out MECs is highly responsive to induction by growth factors such as TGFα and FGFs when compared to MECs from littermate controls. Hyper-proliferative phenotype has been implicated as one of the key factors for breast cancer initiation as well as progression. Overall, our work describes a direct evidence for the role of NMI as a key regulator of mammary epithelial cell proliferation. Citation Format: Hawley C. Pruitt, Brandon J. Metge, Sarah K. Bailey, Lalita A. Shevde, Rajeev S. Samant. N-Myc and STAT Interactor knock out in the mammary epithelium prompts hyper-proliferation. [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 657.

Abstract 4010: MicroRNA regulator of NMI: implications for breast cancer

Recent investigations by our laboratory have demonstrated that NMI expression is down-regulated in patient breast tumors, especially those displaying invasive/metastatic features. In breast cancer and melanoma cell lines, restored expression of NMI suppresses tumor growth. Furthermore, NMI negatively impacts TGFβ-driven epithelial-mesenchymal-transition (EMT) and alsometastatic invasion of breast cancer. Thus the mechanisms that regulate NMI expression are of potential interest to understand the etiology of breast tumor progression, invasion, metastasis and to identify potentially novel therapeutic targets. MicroRNAs are critical regulators of gene expression. MicroRNA target prediction searches revealed that the NMI transcript could be targeted by the miR-29 family, a group of miRNAs with multiple roles in diverse cancer types. We hypothesized that miR-29 will negatively regulate NMI expression. Luciferase reporter assays using pMIR-REPORT TM confirmed the projected interaction of miR-29 in the seed region of the 3′ UTR of NMI. Manipulation of miR-29 levels in breast cancer cells by overexpression or silencing showed its inverse relationship with NMI expression. Invasive mesenchymal-like breast cancer cell lines showed elevated endogenous levels of miR-29 relative to tumorigenic/non-metastatic (epithelial like) breast cancer cell lines. Moreover, transient overexpression of miR-29 enhanced breast cancer cell invasion as measured by modified Boyden chamber assay and silencing miR-29 using anti-miRs decreased invasion. Investigations on the impact of miR-29 modulation on EMT revealed that increasing miR-29 levels in epithelial-like breast cancer cell lines caused 3D morphological changes indicative of invasive mesenchymal-like growth, while decreasing miR-29 levels from mesenchymal-like cell types using anti-miRs reversed the phenotype and the growth pattern resembled acinar structures. The significance of our observation was realized when we compared RNA isolated from patient derived breast cancer tumors and compared with RNA from matched adjacent normal breast specimens. Analysis using the McNemar9s test showed a strong, inverse relationship between the expression of NMI and the miR-29. We contend that aberrant miR-29 expression may account for reduced NMI in some invasive breast cancers. Citation Format: Hawley C. Pruitt, Jack W. Rostas III, Brandon J. Metge, Daniel J. Devine, Sarah K. Bailey, Lalita A. Shevde, Rajeev Samant. MicroRNA regulator of NMI: implications for breast 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 4010. doi:10.1158/1538-7445.AM2014-4010