Dormant Breast Cancer Research Articles

Combined SFK/MEK inhibition prevents metastatic outgrowth of dormant tumor cells

Breast cancer (BC) can recur as metastatic disease many years after primary tumor removal, suggesting that disseminated tumor cells survive for extended periods in a dormant state that is refractory to conventional therapies. We have previously shown that altering the tumor microenvironment through fibrosis with collagen and fibronectin deposition can trigger tumor cells to switch from a dormant to a proliferative state. Here, we used an in vivo preclinical model and a 3D in vitro model of dormancy to evaluate the role of Src family kinase (SFK) in regulating this dormant-to-proliferative switch. We found that pharmacological inhibition of SFK signaling or Src knockdown results in the nuclear localization of cyclin-dependent kinase inhibitor p27 and prevents the proliferative outbreak of dormant BC cells and metastatic lesion formation; however, SFK inhibition did not kill dormant cells. Dormant cell proliferation also required ERK1/2 activation. Combination treatment of cells undergoing the dormant-to-proliferative switch with the Src inhibitor (AZD0530) and MEK1/2 inhibitor (AZD6244) induced apoptosis in a large fraction of the dormant cells and delayed metastatic outgrowth, neither of which was observed with either inhibitor alone. Thus, targeting Src prevents the proliferative response of dormant cells to external stimuli, but requires MEK1/2 inhibition to suppress their survival. These data indicate that treatments targeting Src in combination with MEK1/2 may prevent BC recurrence.

Abstract 1094: Breast cancer metastatic competency in the hepatic niche is driven by E-cadherin re-expression.

Abstract Nearly half of breast cancer metastases will become clinically evident 5 or more years after the cancer was seemingly ablated. This implies that metastatic cancer cells survived over this extended timeframe without emerging as detectable nodules. Breast cancer metastatic dormancy has yet to be universally defined, and is not well understood as a potential means by which clinically evident metastases emerge after being undetected for years (or decades) from the primary tumor diagnosis. Various mechanistic and microenvironmental factors may work independently, collaboratively (or both) in order to allow a dormant metastatic breast cancer cell (or small cluster) to survive long term. At least one tumor dormancy hallmark is likely required: that of E-cadherin re-expression in order to form heterotypic connections between the carcinoma cells and the liver parenchyma. This re-expression is known as the mesenchymal-to-epithelial-reverting-transition (MErT). This re-expression provides not only survival signals to the breast cancer cells but may also establish cell polarity and protection from autocrine signaling (or extracellular factors) that otherwise may prompt the breast cancer cells to initiate metastatic outgrowth. The purpose of this study is to determine E-cadherin's function in the establishment of breast cancer metastases to the liver. The MDA-MB-231 human breast cancer cell line, that can undergo induced MErT ex vivo, was transfected with either exogenously expressing E-cadherin or shRNA to E-cadherin. Xenograft splenic injections of these cell line variants into mice were performed and tumors allowed to grow for 30-60 days until sacrifice. Immunohistochemistry was performed on the liver and splenic tissue sections in order to assess the number and size of metastatic depostis. Preliminary data demonstrate that cells expressing exogenously encoded E-cadherin developed large splenic tumors with fewer to no liver metastases. MDA-MB-231 wild type cells spawned liver metastases about half the time. In order to more fully probe E-cadherin's role in metastatic competency experiments are underway to develop a doxycycline-inducible E-cadherin bicistronic vector allowing for the down-regulation of E-cadherin in the primary tumor and the re-expression in the metastatic niche. Subsequent immunohistochemistry will determine the metastatic E-cadherin status across these MDA-MB-231 cell variants. These results will provide insights into the role of MErT in metastatic establishment. Citation Format: Donald P. Taylor. Breast cancer metastatic competency in the hepatic niche is driven by E-cadherin re-expression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1094. doi:10.1158/1538-7445.AM2013-1094

Abstract 1093: The role of cellular adhesion in escaping prostate cancer tumor dormancy.

Abstract Dissemination of prostate cancer (PCa) cells to the bone marrow appears to be an early event in the PCa disease process. In some patients, these disseminated tumor cells (DTC) proliferate to form active metastases after a prolonged period of undetectable disease following initial treatment. This time period that precedes recurrence is often referred to as tumor dormancy. Prevention of PCa recurrence would present a significant clinical advantage, as patients with bone metastases ultimately stop responding to second line therapies and eventually succumb to the disease. Understanding the biology of tumor dormancy and the mechanism(s) of release from dormancy will allow us to determine which patients may recur and identify possible targets for therapy to prevent disease recurrence. The microenvironment significantly influences tumor cell proliferation and the formation of metastases. With respect to dormancy, cues from the microenvironment have been shown to regulate integrin β1 signaling in breast and epidermoid carcinoma to promote cell proliferation. However, mechanisms of PCa dormancy remain unknown. Additionally, PCa is a heterogeneous disease, further complicating the identification of mechanisms pertinent to dormancy. Here, we show that 5 different LuCaP PCa xenografts, which appear dormant in vitro, are induced to proliferate through contact with each other and activated bone marrow stroma. Furthermore, this effect can be reproduced by constitutively activating the phosphorylation of myosin light chain II, a downstream effector of integrin β1. It has previously been shown that dormant breast cancer cells lacking integrin β1 signaling experience cell cycle arrest in the G1 phase. Thus, we examined changes in genes implicated in cell cycle progression in LuCaP PCa xenografts with constitutively active myosin light chain kinase (MLCK). We find that cyclin dependent kinase 6 (CDK6) is consistently upregulated in LuCaP xenografts with constitutively active MLCK. Furthermore, the transcription factor E2F4, which is implicated in growth arrest, is consistently downregulated. CDK6 is important in release from G1, and has previously been shown to associate with the androgen receptor, a critical player in PCa growth. Taken together, these data point to a potential mechanism by which PCa cells are released from dormancy to form lethal metastases. Targeting this mechanism could provide powerful new treatment opportunities that could reduce the lethality of PCa. Citation Format: Nazanin S. Ruppender, Sandy Larson, Bryce Lakely, Lisha Brown, Ilsa Coleman, Roger Coleman, Peter Nelson, Eva Corey, Linda Snyder, Robert Vessella, Colm Morrissey. The role of cellular adhesion in escaping prostate cancer tumor dormancy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1093. doi:10.1158/1538-7445.AM2013-1093

Middle Ear Metastasis from Dormant Breast Cancer as the Initial Sign of Disseminated Disease 20 Years after Quadrantectomy

We describe an unusual case of breast cancer metastatic to the middle ear in a 71-year-old woman. The metastasis was the initial sign of disseminated disease 20 years after the patient had undergone a quadrantectomy for her primary disease. Computed tomography (CT) demonstrated the presence of an intratympanic mass with a soft-tissue density that was suggestive of chronic inflammation. The patient underwent a canal-wall-down tympanoplasty. When a brownish mass was found around the ossicles, a mastoidectomy with posterior tympanotomy was carried out. However, exposure of the tumor was insufficient, and therefore the posterior wall of the ear canal had to be removed en bloc. Some tumor was left on the round window membrane so that we would not leave the patient with a total hearing loss. Our case highlights the limitations of CT and magnetic resonance imaging in differentiating inflammatory and neoplastic lesions.

Abstract C93: Liver nonparenchymal cells drive metastatic breast cancer survival but fail to initiate mesenchymal to epithelial reversion

Abstract Nearly half of breast cancer metastases will become clinically evident 5 or more years after the cancer was seemingly ablated. This implies that metastatic cancer cells survived over this extended timeframe without emerging as detectable nodules. Breast cancer metastatic dormancy has yet to be universally defined, and is not well understood as a potential means by which clinically evident metastases emerge after being undetected for years (or decades) from the primary tumor diagnosis. Various mechanistic and microenvironmental factors may work independently, collaboratively (or both) in order to allow a dormant metastatic breast cancer cell (or small cluster) to survive long term. At least one tumor dormancy hallmark is likely required: that of E-cadherin re-expression in order to form heterotypic connections between the carcinoma cells and the liver parenchyma. This re-expression is known as the mesenchymal-to-epithelial-reverting-transition (MErT). This re-expression provides not only survival signals to the breast cancer cells but may also establish cell polarity and protection from autocrine signaling (or extracellular factors) that otherwise may prompt the breast cancer cells to initiate metastatic outgrowth. The purpose of this study was to determine whether non-parenchymal liver cells (NPC) contribute to metastatic competency, initiate mesenchymal to epithelial reverting transition (MErT), and trigger metastatic dormancy. We co-cultured human or rat hepatic isolations with highly metastatic human immortalized breast cancer cells (MDA-MB-231). The hepatocytes, non-parenchymal hepatic supernatant, and liver sinusoidal endothelial cell enriched populations were independently cultured with the breast cancer cells in standard monolayer cultures. Because MDA-MB-231 cells do not survive in the hepatocyte culture media alone, we were able to assess the unique properties imparted by the experimental cultures. Confocal immunofluorescent imaging for proliferation and epithelial markers revealed a mesenchymal phenotypic transition only in the hepatocyte/breast cancer cultures. Interestingly the NPC/breast cancer co-cultures promoted cancer cell proliferation (but lower than with hepatocytes included) but did not impart carcinoma epithelial reversion. The liver sinusoidal endothelial cell (LSEC)-enriched co-cultures exhibited much lower breast cancer cell proliferation hinting toward a possible dormancy, though E-cadherin reversion was not detected. These data suggest that perturbations of the parenchymal and non-parenchymal cell ratios in the liver metastatic microenvironment may contribute to metastatic dormancy initiation or stability. Citation Format: Donald P. Taylor, Alan Wells. Liver nonparenchymal cells drive metastatic breast cancer survival but fail to initiate mesenchymal to epithelial reversion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C93.

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.

Does trauma or an intercurrent surgical intervention lead to a short-term increase in breast cancer recurrence rates?

Several lines of evidence suggest that cytokines released as a result of wound healing might reactivate dormant breast cancer metastases. To test this, we examined if accidental trauma or surgery, unrelated to the original cancer, might stimulate the growth of dormant micrometastases and be related to an increase in the recurrence rate in the period after the event. To test this hypothesis, we used data from the ATAC [Arimidex (anastrozole), tamoxifen alone or in combination] trial and coded the data for women who have experienced trauma or surgical procedures unrelated to the cancer. For the initial analysis, we considered recurrences occurring 2-24 months after the traumatic event and also between 2 and 12 months after trauma. In a secondary analysis, we also looked at recurrences in the first 2 months after event. The hazard ratio (HR) for recurrence 2-24 months after event was 0.96 [confidence interval (CI) 0.86-1.07, P = 0.48]; for 2-12 months, it was 0.96 (CI 0.82-1.11, P = 0.58) and for 0-2 months, the HR was 0.87 (CI 0.54-1.38 P = 0.87). Trauma was not associated with an increased rate of breast cancer recurrence in the 24-month window after the event in this large study.

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.

Progestins in Hormone Replacement Therapies Reactivate Cancer Stem Cells in Women With Preexisting Breast Cancers: A Hypothesis

An increased risk of invasive, estrogen receptor-positive (ER+) breast cancer in the combined estrogen plus progestin arm of the Women's Health Initiative menopausal hormone replacement therapy (HRT) trial was in large part responsible for stopping the study prematurely in 2002. Subsequent studies offered several possibilities to explain how addition of progestins to estrogens increases the risk of breast cancer. The authors of this report offer a hypothesis for the increased risk based on their research and that of other investigators. A rare small tumorigenic subpopulation of estrogen receptor-negative, progesterone receptor-negative cancer stem cells is present in experimental ER+, progesterone receptor-positive human breast cancers. Progestins but not estrogens act on the ER+, progesterone receptor-positive differentiated cells (especially in small nascent tumor colonies) to reactivate and revert to the more primitive estrogen receptor-negative, progesterone receptor-negative stem cells without requiring proliferation. The authors propose that a reservoir of occult, undetected, preinvasive breast cancer or dormant breast cancer stem cells is present before the start of estrogen plus progestin therapy in some women. Autopsy data of women over 40 years of age who did not have known breast cancer during life showed that the median prevalence of invasive breast cancer at death was 1.3% (range: 0%–1.8%), and the median prevalence of ductal carcinoma in situ was 8.9% (range: 0%–14%). Estrogens are not involved in the activation process, but once receptors are reacquired, they can act through their mitogenic properties to expand the tumor cell population. The authors believe that improved screening methods are needed to detect occult, possibly dormant, breast cancers before initiation of hormone replacement therapy. If the hypothesis is correct, women with such preexisting malignancies should be excluded from regimens containing systemic progestins.

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

Dormant Wnt-Initiated Mammary Cancer Can Participate in Reconstituting Functional Mammary Glands

The minimal residual disease foci that beget breast cancer relapse after a period of disease dormancy remain uncharacterized despite their enormous clinical importance. To model dormant breast cancer in vivo, we employed a transgenic mouse model in which Wnt1-initiated mammary cancer is doxycycline dependent. After regression of Wnt-dependent cancers, subclinical disease lesions were propagated in vivo using classical tissue recombination techniques. Surprisingly, outgrowths derived from dormant malignant tissue reconstituted morphologically normal ductal trees in wild-type mammary fat pads. Whereas hyperplasia-derived outgrowths remained benign, outgrowths derived from dormant malignancy underwent a morphological transition suggesting single-step transformation following reactivation of Wnt signaling and rapidly yielded invasive mammary tumors. Remarkably, outgrowths derived from dormant malignancy could be serially propagated in vivo and retained the potential to undergo lobuloalveolar differentiation in response to hormones of pregnancy. Matching somatic H-Ras mutations shared by antecedent tumors and descendant mammary ductal outgrowths confirmed their clonal relatedness. Thus, propagation of epithelium that possesses a latent malignant growth program reveals impressive regenerative and developmental potential, supporting the notion that dormant mammary cancers harbor transformed mammary progenitor cells. Our results define an experimental paradigm for elucidating biological properties of dormant malignancy.

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.