Dalit Barkan

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.

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.

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.

β1-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 β1-integrin in regulating the switch from a dormant state to active proliferation and metastasis. Here we discuss the role of β1-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 β1-integrin signaling to target dormant cancer cells and to prevent metastatic recurrence. Clin Cancer Res; 17(23); 7219–23. ©2011 AACR.

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.

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.

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.

Prevention of Conversion of Tumor Dormancy into Proliferative Metastases

Late recurrences of cancer are believed to be due to dormant disease that can persist for long periods following apparently successful treatment of a primary tumor. Clinical tumor dormancy thus creates uncertainty for cancer patients and their physicians, who cannot be certain that their cancer will not recur. We have a poor understanding about which individual patients are at risk for cancer recurrence following a period of tumor dormancy. Thus, in spite of the clinical importance of tumor dormancy, much remains to be learned about the mechanisms responsible for induction of, and release from, dormancy. Here we consider the clinical problem of tumor dormancy and discuss evolving ideas of how tumor dormancy and reinitiation of growth may be regulated, both naturally in the body and therapeutically. A better understanding of mechanisms by which dormancy can be regulated may suggest new therapeutic approaches to either eliminate dormant cancer cells or promote the maintenance of dormancy.

The Role of Fibrosis in Tumor Progression and the Dormant to Proliferative Switch

The extracellular matrix is known to play a pivotal role in normal breast development as well as tumorigenesis and breast cancer progression. Several lines of clinical evidence have associated the presence of fibrotic-like, activated stroma with poor therapeutic response and prognosis in breast cancer patients. Recent evidence suggests that extracellular changes are requisite for the formation of a pre-metastatic niche that provides a permissive environment for disseminated breast cancer cells to survive and proliferate. It is also thought that in the absence of favorable environmental cues at a metastatic site, disseminated tumor cells can be maintained in a dormant, metabolically active state until they encounter or modulate their surroundings into an environment that supports their proliferation. We have shown in vivo that the induction of lung fibrosis via adenoviral instillation of TGFs, which results in collagen-I accumulation, can induce the proliferation of an otherwise dormant breast cancer cell line (D2.0R). We have recapitulated this dormant-to-proliferative switch by collagen-I supplementation in a three dimensional in vitro model of dormancy, suggesting that collagen-I is a major contributor to the overtly proliferative integrin β1-dependent state of the D2.0R cells in fibrotic lungs. This work has highlighted the importance of the integrin β1 pathway and its downstream effectors as principal players in sensing microenvironmental changes by dormant breast cells and activating pro-proliferative pathways resulting in overt metastases.