Assessing a Novel 3D Assay System for Drug Screening against OS Metastasis.

Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant chemotherapy, pulmonary metastasis is common and is the primary cause of morbidity. To improve outcomes for patients, we have developed and optimized a phenotypic screen for drugs that may target OS disseminated tumor cells (DTCs) and inhibit their metastatic outbreak rather than merely screening for cytotoxic activity against proliferating cells, as is commonly conducted in conventional drug discovery approaches. We report on the validation of a previously described 3D reconstituted basement membrane extract (3D BME) model system for tumor dormancy and metastatic outgrowth adapted to clonal pairs of high and low metastatic OS cells. A post-hoc validation of the assay was possible by comparing the activity of a drug in our assay with early evidence of activity in human OS clinical trials (regorafenib and saracatinib). In this validation, we found concordance between our assay and human clinical trial experience We then explored an approved veterinary small molecule inhibitor of Janus kinase-1 (oclacitinib) as a potential drug candidate to take advantage of the high prevalence of OS in pet dogs and its translational value to humans. Despite the biological rationale, we found no evidence to support the use of oclacitinib as an antimetastatic agent in OS. The findings support our 3D BME assay as a highly efficient method to examine drugs for activity in targeting OS DTCs.

Novel mechanism for OSM-promoted extracellular matrix remodeling in breast cancer: LOXL2 upregulation and subsequent ECM alignment.

BACKGROUND: Invasive ductal carcinoma (IDC) is a serious problem for patients as it metastasizes, decreasing 5-year patient survival from > 95 to ~ 27%. The breast tumor microenvironment (TME) is often saturated with proinflammatory cytokines, such as oncostatin M (OSM), which promote epithelial-to-mesenchymal transitions (EMT) in IDC and increased metastasis. The extracellular matrix (ECM) also plays an important role in promoting invasive and metastatic potential of IDC. Specifically, the reorganization and alignment of collagen fibers in stromal ECM leads to directed tumor cell motility, which promotes metastasis. Lysyl oxidase like-2 (LOXL2) catalyzes ECM remodeling by crosslinking of collagen I in the ECM. We propose a novel mechanism whereby OSM induces LOXL2 expression, mediating stromal ECM remodeling of the breast TME. METHODS: Bioinformatics was utilized to determine survival and gene correlation in patients. IDC cell lines were treated with OSM (also IL-6, LIF, and IL-1ß) and analyzed for LOXL2 expression by qRT-PCR and immunolabelling techniques. Collagen I contraction assays, 3D invasion assays, and confocal microscopy were performed with and without LOXL2 inhibition to determine the impact of OSM-induced LOXL2 on the ECM. RESULTS: Our studies demonstrate that IDC patients with high LOXL2 and OSM co-expression had worse rates of metastasis-free survival than those with high levels of either, individually, and LOXL2 expression is positively correlated to OSM/OSM receptor (OSMR) expression in IDC patients. Furthermore, human IDC cells treated with OSM resulted in a significant increase in LOXL2 mRNA, which led to upregulated protein expression of secreted, glycosylated, and enzymatically active LOXL2. The expression of LOXL2 in IDC cells did not affect OSM-promoted EMT, and LOXL2 was localized to the cytoplasm and/or secreted. OSM-induced LOXL2 promoted an increase in ECM collagen I fiber crosslinking, which led to significant fiber alignment between cells and increased IDC cell invasion. CONCLUSIONS: Aligned collagen fibers in the ECM provide pathways for tumor cells to migrate more easily through the stroma to nearby vasculature and tissue. These results provide a new paradigm through which proinflammatory cytokine OSM promotes tumor progression. Understanding the nuances in IDC metastasis will lead to better potential therapeutics to combat against the possibility.

Innovative Approaches in the Battle Against Cancer Recurrence: Novel Strategies to Combat Dormant Disseminated Tumor Cells.

Cancer recurrence remains a great fear for many cancer survivors following their initial, apparently successful, therapy. Despite significant improvement in the overall survival of many types of cancer, metastasis accounts for ~90% of all cancer mortality. There is a growing understanding that future therapeutic practices must accommodate this unmet medical need in preventing metastatic recurrence. Accumulating evidence supports dormant disseminated tumor cells (DTCs) as a source of cancer recurrence and recognizes the need for novel strategies to target these tumor cells. This review presents strategies to target dormant quiescent DTCs that reside at secondary sites. These strategies aim to prevent recurrence by maintaining dormant DTCs at bay, or eradicating them. Various approaches are presented, including: reinforcing the niche where dormant DTCs reside in order to keep dormant DTCs at bay; promoting cell intrinsic mechanisms to induce dormancy; preventing the engagement of dormant DTCs with their supportive niche in order to prevent their reactivation; targeting cell-intrinsic mechanisms mediating long-term survival of dormant DTCs; sensitizing dormant DTCs to chemotherapy treatments; and, inhibiting the immune evasion of dormant DTCs, leading to their demise. Various therapeutic approaches, some of which utilize drugs that are already approved, or have been tested in clinical trials and may be considered for repurposing, will be discussed. In addition, clinical evidence for the presence of dormant DTCs will be reviewed, along with potential prognostic biomarkers to enable the identification and stratification of patients who are at high risk of recurrence, and who could benefit from novel dormant DTCs targeting therapies. Finally, we will address the shortcomings of current trial designs for determining activity against dormant DTCs and provide novel approaches.

A small-molecule ARTS mimetic promotes apoptosis through degradation of both XIAP and Bcl-2.

Many human cancers over-express B cell lymphoma 2 (Bcl-2) or X-linked inhibitor of apoptosis (IAP) proteins to evade cell death. The pro-apoptotic ARTS (Sept4_i2) protein binds directly to both Bcl-2 and XIAP and promotes apoptosis by stimulating their degradation via the ubiquitin-proteasome system (UPS). Here we describe a small molecule, A4, that mimics the function of ARTS. Microscale thermophoresis assays showed that A4 binds XIAP, but not cellular inhibitor of apoptosis protein 1 (cIAP1). A4 binds to a distinct ARTS binding pocket in the XIAP-BIR3 (baculoviral IAP repeat 3) domain. Like ARTS, A4 stimulated poly-ubiquitylation and UPS-mediated degradation of XIAP and Bcl-2, but not cIAP1, resulting in caspase-9 and -3 activation and apoptosis. In addition, over-expression of XIAP rescued HeLa cells from A4-induced apoptosis, consistent with the idea that A4 kills by antagonizing XIAP. On the other hand, treatment with the SMAC-mimetic Birinapant induced secretion of tumour necrosis factor-α (TNFα) and killed ~50% of SKOV-3 cells, and addition of A4 to Birinapant-treated cells significantly reduced secretion of TNFα and blocked Birinapant-induced apoptosis. This suggests that A4 acts by specifically targeting XIAP. The effect of A4 was selective as peripheral blood mononuclear cells and normal human breast epithelial cells were unaffected. Furthermore, proteome analysis revealed that cancer cell lines with high levels of XIAP were particularly sensitive to the killing effect of A4. These results provide proof of concept that the ARTS binding site in XIAP is "druggable". A4 represents a novel class of dual-targeting compounds stimulating apoptosis by UPS-mediated degradation of important anti-apoptotic oncogenes.

Dormant disseminated tumor cells and cancer stem/progenitor-like cells: Similarities and opportunities.

Distant recurrences occurring years after removal of the primary tumor arise from disseminated tumor cells (DTCs) that lie dormant (quiescent/asymptomatic) until they emerge to overt metastases. These quiescent DTCs are resistant to conventional treatments. Hence, to date there is no available treatment which targets dormant DTCs before they form overt metastases. Therefore, understanding the biology of dormant DTCs and the mechanisms of their reactivation is vital in our pursuit to develop therapies to prevent cancer from ever recurring. This review will address the striking similarities between the biology of DTCs and the biology of cancer stem cells (CSCs) or CSC-like cells including cancer progenitor-like cells. These similarities are related to intrinsic mechanisms of survival and quiescence, and their cross-talk with mediators, produced in their surrounding niches that may support either dormancy or outgrowth. Unraveling these similarities may provide us with exciting opportunities to either mitigate the survival of residing dormant DTCs/CSCs or maintain them in a dormant state. Whether the stemness properties of CSCs/cancer progenitor-like cells already comprising the recurring tumor can be exploited in order to differentiate them, and thus promote their dormancy, will be explored as well. Overall, these emerging concepts may provide us with new opportunities to prevent lethal recurrences.

IFN-ß is a macrophage-derived effector cytokine facilitating the resolution of bacterial inflammation.

The uptake of apoptotic polymorphonuclear cells (PMN) by macrophages is critical for timely resolution of inflammation. High-burden uptake of apoptotic cells is associated with loss of phagocytosis in resolution phase macrophages. Here, using a transcriptomic analysis of macrophage subsets, we show that non-phagocytic resolution phase macrophages express a distinct IFN-ß-related gene signature in mice. We also report elevated levels of IFN-ß in peritoneal and broncho-alveolar exudates in mice during the resolution of peritonitis and pneumonia, respectively. Elimination of endogenous IFN-ß impairs, whereas treatment with exogenous IFN-ß enhances, bacterial clearance, PMN apoptosis, efferocytosis and macrophage reprogramming. STAT3 signalling in response to IFN-ß promotes apoptosis of human PMNs. Finally, uptake of apoptotic cells promotes loss of phagocytic capacity in macrophages alongside decreased surface expression of efferocytic receptors in vivo. Collectively, these results identify IFN-ß produced by resolution phase macrophages as an effector cytokine in resolving bacterial inflammation.

EMT and Stemness in Tumor Dormancy and Outgrowth: Are They Intertwined Processes?

Metastases are the major cause of cancer patients' mortality and can occur years and even decades following apparently successful treatment of the primary tumor. Early dissemination of cancer cells, followed by a protracted period of dormancy at distant sites, has been recently recognized as the clinical explanation for this very-long latency. The mechanisms that govern tumor dormancy at distant sites and their reactivation to proliferating metastases are just beginning to be unraveled. Tumor cells, that survive the immune surveillance and hemodynamic forces along their journey in the circulation and successfully colonize and adopt to the new and "hostile" microenvironment and survive in a quiescent dormant state for years before emerging to proliferative state, must display high plasticity. Here we will discuss whether the plasticity of dormant tumor cells is required for their long-term survival and outgrowth. Specifically, we will focus on whether epithelial mesenchymal transition and acquisition of stem-like properties can dictate their quiescent and or their proliferative fate. Deeper understanding of these intertwining processes may facilitate in the future the development of novel therapies.

Soluble Mediators Produced by Pro-Resolving Macrophages Inhibit Angiogenesis.

Different subtypes of macrophages have been shown to participate in different stages of inflammation and tissue repair. In the late stage of tissue repair, the macrophages, following their engulfment of apoptotic neutrophils, acquire a new phenotype termed alternatively activated macrophages. These macrophages produce growth factors, such as vascular endothelial growth factor (VEGF), that facilitate the angiogenic response as part of tissue restoration. Then, in the later stages of tissue healing, capillary regression takes place. It is presently unknown whether macrophages play an antiangiogenic role in the final stages of tissue repair. Here, we examined whether soluble mediators secreted by pro-resolving CD11blow macrophages (Mres) inhibit angiogenesis in the context of the resolution of tissue repair. Our findings indicate that soluble mediators produced by ex vivo generated Mres (CM-Mres) attenuate angiogenesis in vitro by inhibiting human umbilical vein endothelial cell (HUVEC) proliferation by lowering their cyclin D1 expression. In addition, CM-Mres lowered HUVEC survival by inducing caspase 3/7 activation, and also inhibited VEGFR2 activation via VEGF. HUVEC migration and differentiation to tubular-like structure was also inhibited by CM-Mres. Similarly, CM-Mres significantly inhibited neovascularization as depicted ex vivo by utilizing the rat aorta ring assay and in vivo by utilizing the chick chorioallantoic membrane assay. Notably endostatin, which was shown previously to exert its antiangiogenic effect by inhibiting proliferation, survival, motility, and morphogenesis of endothelial cells via inhibition of VEGFR2 activation, is produced by Mres. Taken together, our results suggest that a specialized subset of macrophages that appear during the resolution of inflammation can produce antiangiogenic mediators, such as endostatin. These mediators can halt angiogenesis, thereby restoring tissue structure.

'Normalizing' the malignant phenotype of luminal breast cancer cells via alpha(v)beta(3)-integrin.

Reestablishing tissue organization of breast cancer cells into acini was previously shown to override their malignant phenotype. In our study, we demonstrate that alpha(v)beta(3) integrin (Int-αvß3), previously shown to play a role in cancer progression, promoted differentiation and growth arrest of organoids derived from luminal A breast cancer cells grown in their relevant three-dimensional microenvironment. These organoids differentiated into normal-like acini resembling a benign stage of breast tissue. Likewise, we demonstrate that Int-αvß3 is selectively expressed in the epithelium of the benign stage of breast tissues, and is lost during the early stages of luminal A breast cancer progression. Notably, the organoids' reversion into normal-like acini was mediated by cancer luminal progenitor-like cells expressing both EpCAMhighCD49flowCD24+ and Int-αvß3. Furthermore, downregulation of Notch4 expression and downstream signaling was shown to mediate Int-αvß3-induced reversion. Intriguingly, when luminal A breast cancer cells expressing Int-αvß3 were injected into a humanized mouse model, differentiated tumors developed when compared with that generated by control cells. Hence, our data suggest that promoting differentiation of luminal A breast cancer cells by signaling emanating from Int-αvß3 can potentially promote 'normalization' of their malignant phenotype and may prevent the malignant cells from progressing.

Dormant tumor cells expressing LOXL2 acquire a stem-like phenotype mediating their transition to proliferative growth.

Recurrence of breast cancer disease years after treatment appears to arise from disseminated dormant tumor cells (DTC). The mechanisms underlying the outgrowth of DTC remain largely unknown. Here we demonstrate that dormant MCF-7 cells expressing LOXL2 acquire a cancer stem cell (CSC)-like phenotype, mediating their outgrowth in the 3D BME system that models tumor dormancy and outgrowth. Similarly, MCF-7-LOXL2 cells colonizing the lung transitioned from dormancy to metastatic outgrowth whereas MCF-7 cells remained dormant. Notably, epithelial to mesenchymal transition (EMT) of MCF-7-LOXL2 cells was required for their CSC-like properties and their transition to metastatic outgrowth. These findings were further supported by clinical data demonstrating that increase in LOXL2 mRNA levels correlates with increase in the mRNA levels of EMT and stem cells markers, and is also associated with decrease in relapse free survival of breast cancer patients. Notably, conditional hypoxia induced expression of endogenous LOXL2 in MCF-7 cells promoted EMT and the acquisition of a CSC-like phenotype, while knockdown of LOXL2 inhibited this transition. Overall, our results demonstrate that expression of LOXL2 endowed DTC with CSC-like phenotype driving their transition to metastatic outgrowth and this stem-like phenotype is dependent on EMT that can be driven by the tumor microenvironment.

Non-steroidal anti-inflammatory drugs target the pro-tumorigenic extracellular matrix of the postpartum mammary gland.

Breast cancer patients diagnosed postpartum have poor prognosis. The postpartum mammary gland undergoes tissue regression to return to the pre-pregnant state. This involution is characterized by wound healing programs known to be tumor promotional in other contexts. Previous studies have shown that mammary extracellular matrix (ECM) from nulliparous rats has tumor suppressive attributes, while mammary ECM from involuting mammary glands is promotional. In models of pregnancy-associated breast cancer, non-steroidal anti-inflammatory drug (NSAID) treatment targeted to postpartum involution inhibits tumor progression, in part by suppressing COX-2 dependent collagen deposition. Because mammary ECM proteins are coordinately regulated, NSAID treatment is anticipated to result in additional protective changes in the mammary extracellular matrix. Here, systemic NSAID treatment was utilized during postpartum involution to reduce mammary COX-2 activity. ECM was isolated from actively involuting glands of rats treated with NSAIDs and compared to ECM isolated from control-involution and nulliparous rats in 3D cell culture and xenograft assays. Compositional changes in ECM between groups were identified by proteomics. In four distinct 3D culture assays, normal and transformed mammary epithelial cells plated in NSAID-involution ECM, phenocopied cells plated in ECM from nulliparous rats rather than ECM from control-involution rats. Tumor cells mixed with NSAID-involution ECM and injected orthotopically in mice formed smaller tumors than cells mixed with control-involution ECM. Proteomic analyses identified and 3D culture assays implicated the ECM protein tenascin-C as a potential mediator of tumor progression during involution that is decreased by NSAID treatment. In summary, NSAID treatment decreases tumor-promotional attributes of postpartum involution mammary ECM.

ß1-integrin: a potential therapeutic target in the battle against cancer recurrence.

Primary cancer treatment, involving both local and often systemic adjuvant therapy, is often successful, especially if the cancer is detected at an early stage of progression. However, for some patients, the cancer may recur either locally or as distant metastases, in some cases many years after apparently successful primary treatment. Significant tumor dormancy has been documented in several cancers, such as breast, melanoma, and renal cancer. Tumor dormancy has long been recognized as an important problem in management of cancer patients. Recent work has clarified biologic aspects of tumor dormancy and has shown that dormant tumor cells may be resistant to cytotoxic chemotherapy and radiation. This work has led to recognition of a key role for ß1-integrin in regulating the switch from a dormant state to active proliferation and metastasis. Here we discuss the role of ß1-integrin and its signaling partners in regulating the dormant phenotype. We also consider possible therapeutic approaches, such as small molecules or antibodies (ATN-161, volociximab, and JSM6427), directed against ß1-integrin signaling to target dormant cancer cells and to prevent metastatic recurrence.

An in vitro system to study tumor dormancy and the switch to metastatic growth.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time. The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression and reside growth-arrested in the patients' bone marrow, blood and lymph nodes. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems. In vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo. We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

Metastatic growth from dormant cells induced by a col-I-enriched fibrotic environment.

Breast cancer that recurs as metastatic disease many years after primary tumor resection and adjuvant therapy seems to arise from tumor cells that disseminated early in the course of disease but did not develop into clinically apparent lesions. These long-term surviving, disseminated tumor cells maintain a state of dormancy, but may be triggered to proliferate through largely unknown factors. We now show that the induction of fibrosis, associated with deposition of type I collagen (Col-I) in the in vivo metastatic microenvironment, induces dormant D2.0R cells to form proliferative metastatic lesions through beta1-integrin signaling. In vitro studies using a three-dimensional culture system modeling dormancy showed that Col-I induces quiescent D2.0R cells to proliferate through beta1-integrin activation of SRC and focal adhesion kinase, leading to extracellular signal-regulated kinase (ERK)-dependent myosin light chain phosphorylation by myosin light chain kinase and actin stress fiber formation. Blocking beta1-integrin, Src, ERK, or myosin light chain kinase by short hairpin RNA or pharmacologic approaches inhibited Col-I-induced activation of this signaling cascade, cytoskeletal reorganization, and proliferation. These findings show that fibrosis with Col-I enrichment at the metastatic site may be a critical determinant of cytoskeletal reorganization in dormant tumor cells, leading to their transition from dormancy to metastatic growth. Thus, inhibiting Col-I production, its interaction with beta1-integrin, and downstream signaling of beta1-integrin may be important strategies for preventing or treating recurrent metastatic disease.

Extracellular matrix: a gatekeeper in the transition from dormancy to metastatic growth.

Metastases can develop after apparently successful treatment of a primary tumour, sometimes following a period of tumour dormancy that can last for years. However, factors that regulate metastatic tumour dormancy remain poorly understood. Here we review the potential contribution of interactions between tumour cells and the microenvironment in metastatic sites, in regulating tumour dormancy vs. metastatic growth. We focus particularly on the potential role of the extracellular matrix (ECM) in regulating maintenance and release from dormancy. Tumour cells that fail to properly adhere to the ECM may enter a state of dormancy. The molecular and physical composition of the ECM can be affected by tumour cells themselves, as well as multiple stromal cell types. The roles of integrins, fibronectin, and collagen are discussed, as are factors that can change the ECM. A better understanding of the molecular details of the crosstalk between tumour cells and the ECM in secondary sites, and how these regulate the dormant state, may lead to improved therapeutic strategies to induce or maintain disseminated tumour cells in a dormant state, or alternatively to successfully eradicate dormant cells.

Inhibition of metastatic outgrowth from single dormant tumor cells by targeting the cytoskeleton.

Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in three-dimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin beta1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin beta1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin beta1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch.

Leptin induces ovulation in GnRH-deficient mice.

Leptin-deficient ob/ob mice have reduced gonadotropin-releasing hormone (GnRH) secretion, leading to gonadotropin deficiencies, hypogonadism, and anovulation, which are completely reversed following leptin administration. To determine whether the role of leptin in ovulation is mediated exclusively through GnRH, we studied leptin's action in GnRH-deficient (hpg) mice, as well as ob/ob mice and normal, prepubertal mice in which the GnRH axis was blocked with antide. Following pretreatment with pregnant mare serum gonadotropin, leptin induced ovulation in all three mouse models. Unlike mature normal mice, these ovulations were not triggered by a luteinizing hormone (LH) surge, as demonstrated by lack of increase in its surrogate marker progesterone. Rather, leptin induced hyperemia and leakage in the follicle, as well as the proteinase ADAMTS-1 (a disintegrin and metalloproteinase with a thrombospondin-like motif), which facilitates extrusion of the follicular content. These data show that on top of its role as an inducer of GnRH secretion, leptin may elicit an LH-independent ovulation.