Dormant Breast Cancer Cells Research Articles

BMP/Coco antagonism as a deterministic factor of metastasis dormancy in lung

A recent Cell publication demonstrates that the secreted antagonist of transforming growth factor-beta ligands, Coco, can re-activate previously dormant metastatic breast cancer cells specifically in the lung by inhibiting bone morphogenetic protein (BMP) signaling. The authors provided evidence for a connection between Coco/BMP signaling and molecular and cellular traits of cancer stem cells. Their findings represent a significant advance in our understanding of metastatic dormancy, an extremely important clinical issue that remains understudied. Equally as important, this study also opens interesting avenues for future research.

Implications for breast cancer dormancy in other areas of medicine

Banys et al1 discussed the different viewpoints of breast cancer dormancy; albeit those that are supported by science. This editorial comments on the clinical implications of existing dormant cancer cells, including future decisions to treat with stem cells. Braun et al2 reported that in cases where metastasis of breast cancer occurred in the bone marrow, this resulted in poor prognosis. As discussed by the authors, it is unclear whether breast cancer cells show tropism for an organ, or whether the cells are able to survive in a particular organ because of support from the microenvironment. Perhaps answers to this question might be clearer if a hierarchy of breast cancer cells is developed, beginning with the cancer stem cell. Depending on the cell subset, there might be a different mechanism for dormancy in an organ. The authors discussed two broad methods of dormancy, one in which the cells changed the pattern of cell cycle, and the other, in which the micrometastasis was caused by a balance between cancer cell proliferation and cell death. Each broad view is likely to have many different subdivisions to explain dormancy. For example, the changes in cytokine production by the cancer cells within a distant site could be responsible for the change in cell cycle of the breast cancer cells. In other mechanisms, gap junctional intercellular communication can be established with the stromal cells within the microenvironment.3 Banys et al1 discussed the premetastatic niche caused by fibronectin for the recruitment of bone marrow cells. The occurrence of such a mechanism may provide insights into the reason for breast cancer to be attracted to the bone marrow and in particular, close to the endosteum, where the hematopoietic stromal cells produce extracellular matrix, such as fibronectin. In bone marrow, the supporting cells are already present. This might partly explain why the bone marrow attracts breast cancer cells, and facilitates the cells’ existence. The existence of the cancer cells among extracellular matrices such as fibronectin within the hematopoietic niche, raises an important argument about whether bone invasion is an opportunistic process. Specifically, if premetastatic sites can be established by fibronectin for the attraction of bone marrow cells, this argues in favor of the environment as a preference for dormancy, rather than the organ. This will then support the microenvironment rather than the organ, as the limiting milieu for dormancy. Although Banys et al1 discussed the mutual relationship between the two methods of dormancy, it is possible that the mechanisms of dormancy might be exclusive, and that each will depend on the subset of cancer cells with distinct interactions with cells and factors within the microenvironment. A comment is required on the section regarding circulating tumor cells.1 The review discussed the different findings regarding the circulating tumor cells. The persistent argument for the benefit of removing lymph nodes in breast cancer patients is inclusive of the function of circulating tumor cells.4 The experimental and clinical evidence support a subset of stem cells in breast and other cancers. The authors stated 0.01% of circulating breast cancer cells can produce a single tumor metastasis. This relatively small subset of tumor initiated breast cancer cells is consistent with the low percentages of a stem cell subset reported in tumors. Future challenges include studies that determine whether the subset of circulating tumor cells that form metastasis show properties of stem cells and also, whether these cells resist current treatments. If so, this subset of cell will need to be characterized because this may hold the key to the development of new forms of treatments. It is also important to note whether the circulating cancer cells bypass the lymph nodes. This information would add to ongoing studies to understand how an individual can exist for decades with dormant breast cancer cells.

Disseminated Breast Cancer Cells Acquire a Highly Malignant and Aggressive Metastatic Phenotype during Metastatic Latency in the Bone

BackgroundDisseminated tumor cells (DTCs) in the bone marrow may exist in a dormant state for extended periods of time, maintaining the ability to proliferate upon activation, engraft at new sites, and form detectable metastases. However, understanding of the behavior and biology of dormant breast cancer cells in the bone marrow niche remains limited, as well as their potential involvement in tumor recurrence and metastasis. Therefore, the purpose of this study was to investigate the tumorigenicity and metastatic potential of dormant disseminated breast cancer cells (prior to activation) in the bone marrow.Methodology/Principal FindingsTotal bone marrow, isolated from mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice. As a negative control, bone marrow isolated from non-injected mice was injected into the mammary fat pad of NUDE mice. The resultant tumors were analyzed by immunohistochemistry for expression of epithelial and mesenchymal markers. Mouse lungs, livers, and kidneys were analyzed by H+E staining to detect metastases. The injection of bone marrow isolated from mice previously injected with tumorspheres into the mammary fat pad, resulted in large tumor formation in the mammary fat pad 2 months post-injection. However, the injection of bone marrow isolated from non-injected mice did not result in tumor formation in the mammary fat pad. The DTC-derived tumors exhibited accelerated development of metastatic lesions within the lung, liver and kidney. The resultant tumors and the majority of metastatic lesions within the lung and liver exhibited a mesenchymal-like phenotype.Conclusions/SignificanceDormant DTCs within the bone marrow are highly malignant upon injection into the mammary fat pad, with the accelerated development of metastatic lesions within the lung, liver and kidney. These results suggest the acquisition of a more aggressive phenotype of DTCs during metastatic latency within the bone marrow microenvironment.

A Secreted BMP Inhibitor Mediates Metastatic Colonization in the Lung

Abstract Coco promotes reactivation and metastatic outgrowth of dormant breast cancer cells in the lung.

Recharging with COCO

Reactivation of dormant breast cancer cells at metastatic sites may require these cells to overcome organ-specific suppressive signals in the microenvironment and to activate stem cell-like traits.

The BMP Inhibitor Coco Reactivates Breast Cancer Cells at Lung Metastatic Sites

The mechanistic underpinnings of metastatic dormancy and reactivation are poorly understood. A gain-of-function cDNA screen reveals that Coco, a secreted antagonist of TGF-β ligands, induces dormant breast cancer cells to undergo reactivation in the lung. Mechanistic studies indicate that Coco exerts this effect by blocking lung-derived BMP ligands. Whereas Coco enhances the manifestation of traits associated with cancer stem cells, BMP signaling suppresses it. Coco induces a discrete gene expression signature, which is strongly associated with metastatic relapse to the lung, but not to the bone or brain in patients. Experiments in mouse models suggest that these latter organs contain niches devoid of bioactive BMP. These findings reveal that metastasis-initiating cells need to overcome organ-specific antimetastatic signals in order to undergo reactivation.

Inflammatory Breast Cancer Stem Cells: Contributors to Aggressiveness, Metastatic Spread and Dormancy

Cancer stem cell populations have been identified for several types of cancers and suggest a way for tumor cells to be resistant to therapies. Further, because of the longevity, endurance and replicative potential of cancer cells with stem-like properties, other malignant attributes such as recurrence after long periods of dormancy can also be explained. Inflammatory breast cancer (IBC) is a unique and aggressive form of breast cancer that has a clinical course unlike other forms of breast cancer. The main hallmark of IBC is prolific invasion of the dermal lymphatic vessels by tumor emboli leading to rapid metastasis of the disease. Despite an extremely aggressive treatment approach, the majority of women with IBC present with disease recurrence suggesting the presence of chemo resistant and/or dormant breast cancer cells. Current evidence suggests that IBC tumor emboli contain distinct populations of cells with stem cell-like properties. Thus, specific targeting of these stem cell-like cancer cells may be the key to effectively treating IBC.

Breast cancer stem cells survive periods of farnesyl-transferase inhibitor-induced dormancy by undergoing autophagy.

A cancer stem cell has been defined as a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. These tumor-forming cells could hypothetically originate from stem, progenitor, or differentiated cells. Previously, we have shown that breast cancer cells with low metastatic potential can be induced into a reversible state of dormancy by farnesyl transferase inhibitors (FTIs). Dormancy was induced by changes in RhoA and RhoC GTPases. Specifically, RhoA was found to be hypoactivated while RhoC was hyperactivated. In the current study we demonstrate that these dormant cells also express certain known stem cell markers such as aldehyde dehydrogenase I (ALDHI) and cluster of differentiation 44 (CD44). We also show that autophagy markers Atg5, Atg12, and LC3-B are expressed in these dormant stem cell-like breast cancer cells. Inhibiting autophagy by inhibitor 3-methyladenine (3-MA) blocked the process of autophagy reversing the dormant phenotype. Further, we show that c-jun NH2 terminal kinase (JNK/SAPK) is upregulated in these dormant stem cell-like breast cancer cells and is responsible for increasing autophagy.

Autologous fat transplantation in patients with breast cancer: “silencing” or “fueling” cancer recurrence?

Lipotransfer can be considered a technical revolution in plastic surgery and widely performed for esthetic surgery. Recently the lipofilling has been indicated in breast reconstruction and deformity correction after breast conservative treatment. However, there is lack of understanding concerning the interactions between the potential tumor beds and the lipoaspirates grafts. Current literature underlines the efficacy of the technique as well as its safety. Nevertheless, many experimental studies provide data on the endocrine, paracrine, and autocrine activities of the transplanted fat tissues. Adipocyte, pre-adipocyte and progenitor cell secretions can stimulate angiogenesis and cell growth. The “tumor–stroma interaction” can potentially induce cancer reappearance by “fueling” dormant breast cancer cells in tumor bed. There is lack of translational research that proves this concern in clinical aspect. No study on the effects of lipotransfer on human cancer breast cells in vivo is available. We provide direct and indirect effects of lipotransfer in breast cancer patients, highlighting pro and con related issues. To confirm the safety of lipotransfer in breast cancer patients we need clinical studies with control group based on long term follow up.

Abstract P4-06-10: Altered Activity of RhoA and RhoC GTPases Phosphorylate C-Jun N-Terminal Kinase and Induce Cellular Dormancy in Breast Cancer Cells

Abstract Tumor dormancy has been a very real problem in breast cancer survivors where recurrences are common and usually fatal. Molecular mechanisms involved in this process are often poorly understood and are a “hot-spot” in breast cancer research today. Ongoing efforts in our laboratory has shown that Rho GTPases, specifically RhoA and RhoC, play a significant role in dormancy of breast cancer. We have currently established two in vitro models of breast tumor dormancy — one involving growth of the breast cancer cell line MCF7 on fibronectin and subsequent treatment with basic fibroblast growth factor and the other involving treatment of MCF7 cells with the farnesyl transferase inhibitor FTI-L-744,832. In both these models, we find high levels of active RhoC and almost undetectable levels of active RhoA GTPases in quiescent breast tumor cells. Investigating further downstream we found c-jun N-terminal kinase (JNK) is phosphorylated in these dormant cells but not p38mitogen activated protein kinase(MAPK) or extracellular signalregulated kinase(ERK1/2). On inhibiting JNK with a pharmacological inhibitor specific for JNK, SP600125, we found dormancy could be interrupted and cell proliferation was induced. JNK has been implicated in inducing autophagy in cells by activating autophagy gene Atg5 and Beclin1 (BCN1) [Byun et al., Carcinogenesis, 2009]. Since autophagy can be a mode of survival for the dormant tumor cell [Matthew et al., Cancer Cell, 2006] we explored the possibility of autophagy being induced in these dormant tumor cells via JNK by employing the autophagy marker LC3-II. We found upregulation of the autophagy marker, thus revealing autophagy as a strategy for survival for these tumor cells in unfavorable tumor microenvironment. We are currently investigating the molecular mechanisms underlying the induction of dormancy in breast tumor cells by Rho GTPases through JNK and the dormant tumor survival strategy through autophagy. We believe that targeting these implicated molecules like RhoA and RhoC GTPases, JNK or some of the autophagy genes might yield therapeutic benefits for breast cancer patients in remission and wipe out fears of deadly metastatic recurrences thus prolonging survival. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-10.

Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype

Farnesyl transferase inhibitors (FTIs) were shown to be effective in modulating tumor growth in Ras-transformed tumor cells. Recent studies have focused on Rho GTPases as putative targets of FTI action. Previously, we demonstrated that FTIs were effective in inhibiting the growth and invasiveness of RhoC GTPase-overexpressing inflammatory breast cancer (IBC) cells however, RhoC activity was increased. In this study, we examine the mechanisms of FTI action on breast cancer cells in culture through modulation of RhoC and RhoA GTPases. We found that FTI inhibition of breast cancer cell growth was reversible and resembled what has been described for an in vitro model of tumor cell dormancy. On FTI treatment, levels of active RhoA decreased significantly, whereas levels of active RhoC increased 3.8-fold. We studied the role of these two GTPases in a fibronectin and basic FGF-induced model of breast cancer cell dormancy. Hypoactivation of RhoA and hyperactivation of RhoC were seen to induce morphology and growth changes consistent with tumor cell dormancy in culture. In addition, the JNK/SAPK pathway was induced on FTI treatment. A pharmacologic inhibitor of the JNK/SAPK pathway significantly reduced the number of dormant cells. This study has implications for the use of FTIs as therapeutic agents as well as potential mechanisms for breast cancer cell dormancy.

Breast cancer cell dormancy in bone marrow: potential therapeutic targets within the marrow microenvironment

Breast cancer cell dormancy in bone marrow: potential therapeutic targets within the marrow microenvironment

Cytokeratin 19 regulates endoplasmic reticulum stress and inhibits ERp29 expression via p38 MAPK/XBP-1 signaling in breast cancer cells

Cytokeratin 19 (CK19) is widely used as a biomarker for the detection of disseminated tumor cells in blood and bone marrow, and its positivity is considered as an independent prognostication indicator in cancer patients. However, its role in breast cancer progression remains unknown. We had established a stable CK19-expressing clone in the CK19-negative BT549 human breast cancer cell line and found that CK19 expression in the BT549 cells caused cell cycle arrest, reduced cell motility and increased drug resistance. Further study revealed that CK19 expression regulated endoplasmic reticulum (ER) stress signaling by up-regulating p38/RNA-dependent protein kinase-like ER kinase (PERK)/p-eIF2α and 78 kDa glucose-regulated protein (Bip/GRP78), and down-regulating focal adhesion kinase (FAK). The level of ER protein 29 (ERp29) was shown to be decreased in the CK19-expressing BT549 cells by proteomic analyses and verified by Western blotting and RT-PCR. Pharmacological inhibition of p38 signaling by its specific inhibitor SB203580 or knockdown of p38 and transcription factor XBP-1 by siRNA in BT549/CK19 and MDA-MB-231 cells revealed that p38/XBP-1 signaling negatively regulated ERp29 expression. Our results indicated that CK19 modulates ER stress signaling and contributes to cell survival and dormancy in breast cancer cells.

Rapid Testosterone Cycling and Chemotherapy for Prostate Cancer: A Way Forward or Return to the Past?

Androgen deprivation therapy is the mainstay of treatment for metastatic prostate cancer. Among men with overt metastatic disease, androgen deprivation therapy by either bilateral orchiectomies or chronic treatment with a gonadotropin-releasing hormone agonist achieves responses in the vast majority, with a median response duration is approximately 1 to 2 years. Only rarely does androgen deprivation therapy result in prolonged remission or cure. Docetaxel is the standard first-line chemotherapy for men who develop disease progression despite androgen deprivation therapy. In two randomized controlled trials in men with castrate-resistant prostate cancer, docetaxel improved overall survival compared with treatment with mitoxantrone. For men with castrate-resistant disease, the current standard of care is to continue androgen deprivation therapy indefinitely, based on concerns that testosterone recovery might accelerate disease progression. The strategy of continuous androgen deprivation therapy and the addition of chemotherapy after emergence of hormone-resistant disease contrasts with the current paradigm of sequential hormone therapy and chemotherapy in breast cancer. In metastatic breast cancer, attempts to improve efficacy by combining hormone therapy and chemotherapy began decades ago. Several randomized controlled trials have compared hormone therapy to hormone therapy plus chemotherapy. Most studies report higher response rates for combination therapy; none report a significant improvement in survival. Other randomized controlled trials have compared chemotherapy alone to chemotherapy plus hormone therapy. Most of those studies reported significantly higher response rates for combination therapy but did not carefully evaluate the cumulative response rates for subjects who received sequential chemotherapy followed by hormone therapy at disease progression. None of the studies have reported a significant improvement in overall survival. An Australian study carefully evaluated concurrent and sequential chemotherapy and hormone therapy for metastatic breast cancer. In that study, 339 women with metastatic breast cancer were assigned randomly to one of three groups: tamoxifen, cyclophosphamide and doxorubicin (CA), or concurrent tamoxifen and CA. Women assigned to tamoxifen were treated with salvage chemotherapy at disease progression. The initial response rates to either CA (45%) or concurrent tamoxifen and CA (50%) were significantly higher than tamoxifen alone (22%). With salvage chemotherapy, however, the cumulative response rates were similar for each of the arms. Overall survival was also similar for each of the regimens. The North American Intergroup designed a study (Intergroup 0100/Southwest Oncology Group 8814) in the late 1980s to address the sequencing of hormone therapy and chemotherapy in the adjuvant setting. A total of 1,477 postmenopausal women with estrogen receptor–positive or progesterone receptor–positive nodepositive breast cancer were randomly assigned to tamoxifen alone, tamoxifen plus concurrent cyclophosphamide, doxorubicin, and fluorouracil (CAF), or CAF followed by tamoxifen. The combination of tamoxifen and CAF was superior to tamoxifen alone. Sequential CAF followed by tamoxifen, however, reduced the incidence of recurrence or death by approximately 50% compared with concurrent therapy. Based on these findings, most clinicians have adopted the strategy of administering chemotherapy followed by hormone therapy for the adjuvant treatment of estrogen receptor–positive breast cancer. Notably, other studies in both premenopausal and postmenopausal women have reported no difference between concurrent and sequential tamoxifen and chemotherapy in the adjuvant setting. In preclinical models of breast cancer, estrogens recruit dormant breast cancer cells into the cell cycle. In phase II studies of women with locally advanced or metastatic breast cancer, short-term pretreatment with estrogen before chemotherapy was associated with relatively high response rates. In one study, for example, estrogen priming and chemotherapy were also associated with comparatively long response duration and survival. A randomized controlled trial from the Netherlands, however, demonstrated no benefit from estrogen priming before chemotherapy. In that study, 318 women with stage II/IIIA breast cancer were assigned randomly to fluorouracil, doxorubicin, and cyclophosphamide with or without pretreatment with ethinyl estradiol. After a median follow-up of 6.8 years, there were no significant differences in relapse-free and overall survival between the groups. Similar results were observed in multivariate analyses that adjusted for subject and disease characteristics. The slow growth rate of prostate cancer may partially explain its limited sensitivity to chemotherapy. In the Dunning rat model of prostate cancer, androgen priming induces cell proliferation and increases sensitivity to chemotherapy compared with chemotherapy alone. The strategy of androgen priming to increase the efficacy of chemotherapy in men with prostate cancer was first evaluated more than two decades ago. In 1988, Manni et al reported the results of a small randomized controlled trial of men with castrate-resistant metastatic prostate cancer. A total of 85 men with disease progression despite bilateral orchiectomies were randomly assigned to monthly chemotherapy (CAF) or monthly chemotherapy preceded by three JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 26 NUMBER 18 JUNE 2

Why do some breast cancer cells remain dormant?

Dormant breast cancer cells are a reality that cannot be overlooked. They may stay dormant either after a spread of cancer cells caused by surgery or after being generated by spontaneous or induced mutations in the course of breast gland growth. Some cases are presented in support of both possibilities, followed by a critical appraisal of the factors that may either keep them dormant or later stimulate their growth into a breast cancer.

Flavopiridol Blocks Integrin-Mediated Survival in Dormant Breast Cancer Cells

Breast cancer micrometastases in the bone marrow are resistant to chemotherapy. They can remain dormant for years before some begin to proliferate. We seek to understand survival mechanisms and develop targeted approaches to eliminating these cells. In an in vitro model of dormancy, basic fibroblast growth factor 2 (FGF-2), abundant in the bone marrow, inhibits the growth of well-differentiated cells in the 2- to 10-cell stage and up-regulates integrin alpha(5)beta(1). Through this integrin, cells bind fibronectin, spread out, and acquire a survival advantage, partly through activation of the phosphatidylinositol 3-kinase/Akt pathway. We investigated the effects of Taxotere, flavopiridol, and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and p38 inhibitors on survival of dormant clones and that of flavopiridol on expression of integrins, adhesion strength, and phosphorylation of Akt, ERK 1/2, and p38. Dormant MCF-7 and T-47D cell clones were resistant to Taxotere concentrations 10-fold higher than needed to eliminate growing clones but were almost completely eradicated by 200 nmol/L flavopiridol. Flavopiridol caused a decrease in FGF-2-induced expression of integrins, including alpha(5) and beta(1), and decreased FGF-2-induced specific adhesion to fibronectin. It diminished Akt phosphorylation, but reexpression of active Akt was not sufficient to reverse dormant clone inhibition. Flavopiridol did not affect phosphorylation of ERK 1/2 and p38 but diminished total protein levels. Chemical inhibition of these pathways partially abrogated dormant clone survival. Flavopiridol has pleiotropic effects on key targets involved with survival of dormant breast cancer cells and may represent a useful approach to eliminating cells dependent on multiple signal pathways for survival.

Integrin alpha5beta1 promotes survival of growth-arrested breast cancer cells: an in vitro paradigm for breast cancer dormancy in bone marrow.

The mechanisms of long-term survival of occult breast cancer cells in the bone marrow microenvironment are not known. Using selected bone marrow stromal components with demonstrated roles in promoting growth arrest and survival of breast cancer cells, we reconstituted an in vitro model for dormancy of breast cancer cells in bone marrow. According to this model, basic fibroblast growth factor, a mammary differentiation factor abundant in the bone marrow stroma, induces growth arrest of relatively well-differentiated breast cancer cells, induces a spread appearance, and restricts their survival to fibronectin by up-regulating integrin alpha5beta1. Most of the basic fibroblast growth factor-arrested cells fail to establish optimal ligation to fibronectin and undergo cell death. Cells that do attach to fibronectin, another major constituent of the bone marrow microenvironment, stay alive and growth-arrested for many weeks. Although capable of adhering to other stromal proteins collagen and laminin, dormant cells do not gain a survival advantage from these interactions. Using function-blocking peptides, we show a specific contribution of alpha5beta1-fibronectin interaction in maintaining survival of growth-arrested cells, potentially by negatively modulating apoptotic response via signaling pathways. Blocking of phosphatidylinositol 3'-kinase and Akt inhibits survival of dormant clones, demonstrating this as one of those pathways. Experiments with human bone marrow stroma cocultures confirm the role of fibronectin ligation in maintaining survival of dormant clones.

Addition of granulocyte-colony stimulating factor (G-CSF) to adjuvant treatment may increase survival in patients with operable breast cancer: interaction of G-CSF with dormant micrometastatic breast cancer cells

All cancers are clonal and represent the progeny of a single cell. The unclear point is which clonogenic cells within the tumor clone possess tumor-initiating cell (T-IC) function and are capable of maintaining tumor growth. Stem cells have the ability to divide almost indefinitely. The division can give rise to a new stem cell as well as differentiated cells of the tumor. Breast tumors are comprised of phenotypically diverse populations of breast cancer cells. Among them, the breast cancer stem cell is important for regrowth of tumor and metastasis. Granulocyte-colony stimulating factor (G-CSF) stimulates the pluripotent stem cell beside neutrophil precursors. Breast cancer stem cells which have not been characterized totally may carry the almost identical antigens with hematopoietic stem cell. The dose-intense therapies with the addition of G-CSF in the adjuvant treatment of breast cancer improved clinical outcomes significantly. Presence of micrometastasis in bone marrow of the breast cancer patients is predictor of relapse free survival and important prognostic factor. Actually, breast cancer stem cells in the thousands of micrometastatic cancer cells have the capacity to repopulate and metastasise. We hypothesize that G-CSF use in adjuvant treatment of breast cancer may activate and repopulate these dormant breast cancer stem cells besides its stimulation on blood stem cells. So activated breast cancer stem cells become chemosensitive to cell-cycle specific various chemotherapeutic agents. Improvement in overall survival in operable breast cancer patients having been treated by dose-dense therapies may also be explained by this mechanism.