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The bone is a preferred site for metastatic homing of prostate cancer cells. Once prostate cancer patients develop skeletal metastases, they eventually succumb to the disease; therefore, it is imperative to identify key molecular drivers of this process. This study examines the involvement of protein kinase C epsilon (PKCe), an oncogenic protein that is abnormally overexpressed in human tumor specimens and cell lines, on prostate cancer cell bone metastasis. PC3-ML cells, a highly invasive prostate cancer PC3 derivative with bone metastatic colonization properties, failed to induce skeletal metastatic foci upon inoculation into nude mice when PKCe expression was silenced using shRNA. Interestingly, while PKCe depletion had only marginal effects on the proliferative, adhesive, and migratory capacities of PC3-ML cells in vitro or in the growth of xenografts upon s.c. inoculation, it caused a significant reduction in cell invasiveness. Notably, PKCe was required for transendothelial cell migration (TEM) as well as for the growth of PC3-ML cells in a bone biomimetic environment. At a mechanistic level, PKCe depletion abrogates the expression of IL1β, a cytokine implicated in skeletal metastasis. Taken together, PKCe is a key factor for driving the formation of bone metastasis by prostate cancer cells and is a potential therapeutic target for advanced stages of the disease. Implications: This study uncovers an important new function of PKCe in the dissemination of cancer cells to the bone; thus, highlighting the promising potential of this oncogenic kinase as a therapeutic target for skeletal metastasis. Mol Cancer Res; 13(9); 1336–46. ©2015 AACR . This article is featured in Highlights of This Issue, [p. 1263][1] [1]: /lookup/volpage/13/1263?iss=9

Despite the progress made in the early detection and treatment of prostate adenocarcinoma, the metastatic lesions from this tumor are incurable. We used genome-wide expression analysis of human prostate cancer cells with different metastatic behavior in animal models to reveal that bone-tropic phenotypes upregulate three genes encoding for the cytokine interleukin-1β (IL-1β), the chemokine CXCL6 (GCP-2), and the protease inhibitor elafin (PI3). The Oncomine database revealed that these three genes are significantly upregulated in human prostate cancer versus normal tissue and correlate with Gleason scores ≥7. This correlation was further validated for IL-1β by immunodetection in prostate tissue arrays. Our study also shows that the exogenous overexpression of IL-1β in nonmetastatic cancer cells promotes their growth into large skeletal lesions in mice, whereas its knockdown significantly impairs the bone progression of highly metastatic cells. In addition, IL-1β secreted by metastatic cells induced the overexpression of COX-2 (PTGS2) in human bone mesenchymal cells treated with conditioned media from bone metastatic prostate cancer cells. Finally, we inspected human tissue specimens from skeletal metastases and detected prostate cancer cells positive for both IL-1β and synaptophysin while concurrently lacking prostate-specific antigen (PSA, KLK3) expression. Collectively, these findings indicate that IL-1β supports the skeletal colonization and metastatic progression of prostate cancer cells with an acquired neuroendocrine phenotype. Cancer Res; 73(11); 3297–305. ©2013 AACR.

Cyclin D1b is a splice variant of the cell cycle regulator cyclin D1 and is known to harbor divergent and highly oncogenic functions in human cancer. While cyclin D1b is induced during disease progression in many cancer types, the mechanisms underlying cyclin D1b function remain poorly understood. Herein, cell and human tumor xenograft models of prostate cancer were utilized to resolve the downstream pathways that are required for the protumorigenic functions of cyclin D1b. Specifically, cyclin D1b was found to modulate the expression of a large transcriptional network that cooperates with androgen receptor (AR) signaling to enhance tumor cell growth and invasive potential. Notably, cyclin D1b promoted AR-dependent activation of genes associated with metastatic phenotypes. Further exploration determined that transcriptional induction of SNAI2 (Slug) was essential for cyclin D1b-mediated proliferative and invasive properties, implicating Slug as a critical driver of disease progression. Importantly, cyclin D1b expression highly correlated with that of Slug in clinical samples of advanced disease. In vivo analyses provided strong evidence that Slug enhances both tumor growth and metastatic phenotypes. Collectively, these findings reveal the underpinning mechanisms behind the protumorigenic functions of cyclin D1b and demonstrate that the convergence of the cyclin D1b/AR and Slug pathways results in the activation of processes critical for the promotion of lethal tumor phenotypes.

Recent studies suggest that peroxisome proliferator-activated receptor gamma (PPARγ) agonists may have cancer chemopreventive activity. Other studies have shown that loss of epidermal PPARγ results in enhanced chemical carcinogenesis in mice via unknown mechanisms. However, ultraviolet B (UVB) exposure represents the primary etiological agent for skin cancer formation and the role of PPARγ in photobiology and photocarcinogenesis is unknown. In previous studies, we demonstrated that UVB irradiation of cells results in the formation of oxidized glycerophosphocholines that exhibit PPARγ ligand activity. We therefore hypothesized that PPARγ would prove to be a chemopreventive target in photocarcinogenesis. We first showed that UVB irradiation of mouse skin causes generation of PPARγ agonist species in vivo. We then generated SKH-1 hairless, albino mice deficient in epidermal Pparg (Pparg-/-(epi)) using a cytokeratin 14 driven Cre-LoxP strategy. Using a chronic model of UVB photocarcinogenesis, we next showed that Pparg-/-(epi) mice exhibit an earlier onset of tumor formation, increased tumor burden and tumor progression. Increased tumor burden in Pparg-/-(epi) mice was accompanied by a significant increase in epidermal hyperplasia and p53 positive epidermal cells in surrounding skin lacking tumors. After acute UVB irradiation, Pparg-/-(epi) mice exhibited an augmentation of both UVB-induced Caspase 3/7 activity and inflammation. Increased apoptosis and inflammation was also observed after treatment with the PPARγ antagonist GW9662. With chronic UVB irradiation, Pparg-/-(epi) mice exhibited a sustained increase in erythema and transepidermal water loss relative to wildtype littermates. This suggests that PPARγ agonists could have possible chemopreventive activity in non-melanoma skin cancer.

Metastasis represents by far the most feared complication of prostate carcinoma and is the main cause of death for patients. The skeleton is frequently targeted by disseminated cancer cells and represents the sole site of spread in more than 80% of prostate cancer cases. Compatibility between select malignant phenotypes and the microenvironment of colonized tissues is broadly recognized as the culprit for the organ-tropism of cancer cells. Here, we review our recent studies showing that the expression of platelet-derived growth factor receptor alpha (PDGFRα ) supports the survival and growth of prostate cancer cells in the skeleton and that the soluble fraction of bone marrow activates PDGFRα in a ligand-independent fashion. Finally, we offer pre-clinical evidence that this receptor is a viable target for therapy.

Recent evidence indicates that cancer cells, even in the absence of a primary tumor, recirculate from established secondary lesions to further seed and colonize skeleton and soft tissues, thus expanding metastatic dissemination and precipitating the clinical progression to terminal disease. Recently, we reported that breast cancer cells utilize the chemokine receptor CX3CR1 to exit the blood circulation and lodge to the skeleton of experimental animals. Now, we show that CX3CR1 is overexpressed in human breast tumors and skeletal metastases. To assess the clinical potential of targeting CX3CR1 in breast cancer, a functional role of CX3CR1 in metastatic seeding and progression was first validated using a neutralizing antibody for this receptor and transcriptional suppression by CRISPR interference (CRISPRi). Successively, we synthesized and characterized JMS-17-2, a potent and selective small-molecule antagonist of CX3CR1, which was used in preclinical animal models of seeding and established metastasis. Importantly, counteracting CX3CR1 activation impairs the lodging of circulating tumor cells to the skeleton and soft-tissue organs and also negatively affects further growth of established metastases. Furthermore, nine genes were identified that were similarly altered by JMS-17-2 and CRISPRi and could sustain CX3CR1 prometastatic activity. In conclusion, these data support the drug development of CX3CR1 antagonists, and promoting their clinical use will provide novel and effective tools to prevent or contain the progression of metastatic disease in breast cancer patients. Implications: This work conclusively validates the instrumental role of CX3CR1 in the seeding of circulating cancer cells and is expected to pave the way for pairing novel inhibitors of this receptor with current standards of care for the treatment of breast cancer patients. Mol Cancer Res; 14(6); 518–27. ©2016 AACR.

The molecular mechanisms underlying metastatic dissemination are still not completely understood. We have recently shown that β1 integrin-dependent cell adhesion to fibronectin and signaling is affected by a transmembrane molecule, Trop-2, which is frequently upregulated in human carcinomas. Here, we report that Trop-2 promotes metastatic dissemination of prostate cancer cells in vivo and is abundantly expressed in metastasis from human prostate cancer. We also show here that Trop-2 promotes prostate cancer cell migration on fibronectin, a phenomenon dependent on β1 integrins. Mechanistically, we demonstrate that Trop-2 and the α5β1 integrin associate through their extracellular domains, causing relocalization of α5β1 and the β1-associated molecule talin from focal adhesions to the leading edges. Trop-2 effect is specific as this molecule does not modulate migration on vitronectin, does not associate with the major vitronectin receptor, αvβ3 integrin, and does not affect localization of αvβ3 integrin as well as vinculin in focal adhesions. We show that Trop-2 enhances directional prostate cancer cell migration, through modulation of Rac1 GTPase activity. Finally, we show that Trop-2 induces activation of PAK4, a kinase that has been reported to mediate cancer cell migration. In conclusion, we provide the first evidence that β1 integrin-dependent migratory and metastatic competence of prostate cancer cells is enhanced by Trop-2. Cancer Res; 73(10); 3155–67. ©2013 AACR.

Therapeutic approaches aimed to prevent breast cancer cells from spreading to distant sites are commonly neglected, based on the indication that dissemination from primary tumors can occur early. However, mounting evidence indicates that cancer cells recirculating from established bone lesions could further seed and colonize both skeleton and soft-tissue organs, thus precipitating metastatic dissemination and accelerating disease progression. We have previously shown that breast circulating tumor cells (CTCs) rely on the chemokine receptor CX3CR1 for lodging to the skeleton. Here, we show that this receptor is overexpressed by all four main subtypes of breast adenocarcinoma and also abundantly detected in bone metastatic lesions from breast cancer patients. We have recently synthesized JMS-17-2, a novel small molecule inhibitor of CX3CR1. We tested this compound in pre-clinical animal models of metastases in which human breast cancer cells, labeled with fluorescent and bioluminescent markers, are converted into CTCs by direct grafting in the arterial blood circulation. Pre-incubating cancer cells with JMS-17-2 or treating animals with this compound (10 mg/Kg, i.p.) were equally effective in dramatically decreasing the number of breast CTCs that lodged to the skeleton. These disseminated tumor cells (DTCs) were detected 24 hours after grafting and enumerated by multispectral fluorescence microscopy of sequential bone tissue sections spanning the entire width of tibiae and femora. Notably, when animals were examined at two weeks after grafting, JMS-17-2 completely prevented metastases, indicating that the reduction in number of DTCs directly translates to an impaired tumor growth in the long term. Finally, JMS-17-2 was administered to animals harboring a limited number of small metastases in bone and soft-tissues and monitored by bioluminescence imaging for three weeks before sacrifice. Animals treated with the CX3CR1 antagonist remained at an oligometastatic stage whereas control animals showed a sharp increase in the number of metastatic lesions and overall tumor burden. The work presented here should generate a conceptual shift in the treatment strategies for breast cancer patients, paving the way to approaches aimed to counteract the seeding of additional lesions from existing metastases. We also introduce JMS-17-2 as the first CX3CR1 antagonist and lead compound of a class of potentially new drugs with a novel mechanism of action that could be added to the arsenal of therapies currently used to treat advanced breast adenocarcinoma. Citation Format: Fei Shen, Yun Zhang, Danielle Jernigan, Jie Yan, Fernando Garcia, Olimpia Meucci, Joseph Salvino, Alessandro Fatatis. A small-molecule antagonist of CX3CR1 impairs homing and colonization of breast cancer cells in the skeleton. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B03.

Metabolic reprogramming is an important driver of tumor progression; however, the metabolic regulators of tumor cell motility and metastasis are not understood. Here, we show that tumors maintain energy production under nutrient deprivation through the function of HSP90 chaperones compartmentalized in mitochondria. Using cancer cell lines, we found that mitochondrial HSP90 proteins, including tumor necrosis factor receptor-associated protein-1 (TRAP-1), dampen the activation of the nutrient-sensing AMPK and its substrate UNC-51-like kinase (ULK1), preserve cytoskeletal dynamics, and release the cell motility effector focal adhesion kinase (FAK) from inhibition by the autophagy initiator FIP200. In turn, this results in enhanced tumor cell invasion in low nutrients and metastatic dissemination to bone or liver in disease models in mice. Moreover, we found that phosphorylated ULK1 levels were correlated with shortened overall survival in patients with non-small cell lung cancer. These results demonstrate that mitochondrial HSP90 chaperones, including TRAP-1, overcome metabolic stress and promote tumor cell metastasis by limiting the activation of the nutrient sensor AMPK and preventing autophagy.

Skeletal dissemination is by far the most feared complication of prostate cancer and the main cause of patients’ demise. Today, 85% of patients are diagnosed with prostate cancer in the absence of clinically evident secondary tumors. However, a significant number of these patients will eventually develop the advanced form of the disease and present with skeletal metastases. A definitive, curative treatment for bone metastatic disease is still an unmet clinical need and the current standard of care relies on merely palliative measures.

Advanced prostate cancer (PCa) frequently leads to bone metastasis, a major cause of morbidity and mortality. Limited knowledge of the molecular interactions underpinning the behavior of cancer cells in the bone microenvironment has stymied the development of curative therapies. Signaling between cancer cells and stroma likely plays a crucial role in dictating the fate of disseminated tumor cells (DTCs), and its disruption could interfere with the supportive role of the metastatic niche and thus hamper tumor growth. We previously identified interleukin-1β (IL-1β) as a major mediator of the bone-metastatic behavior of human prostate cancer cells inoculated into the left cardiac ventricle of SCID mice. Here, we show not only that pharmacologic disruption of IL-1β signaling using the IL-1 receptor (IL-1R) antagonist anakinra diminishes the metastatic potential of PCa cells in vivo, but also that IL-1β secreted by PCa cells can prompt bone stromal cells to acquire a carcinoma-associated fibroblast (CAF) phenotype. Emphasizing the role of the stroma, we demonstrate that SCID mice null for IL-1R display diminished formation of bone metastases relative to wild-type when inoculated with highly metastatic IL-1β-expressing PCa cells. In this same model we find that IL-1β supports early survival of DTCs in the bone. Interestingly, while the aggressively-metastatic androgen receptor (AR)-negative PC3-ML cell line expresss high levels of IL-1β, the numerous AR(+) PCa cell lines we examined each lack IL-1β expression. This was recapitulated in human tissue specimens of PCa bone metastases examined by a combination of Laser Capture Microdissection and qPCR. In these lesions, a relevant fraction of PCa cells lack AR transcript and express IL-1β, whereas AR(+) cells were uniformly negative for this cytokine. As AR(+) PCa cells are consistently non-metastatic in our pre-clinical model, we speculated that IL-1β-mediated induction of CAFs in the bone may enable growth of AR(+) PCa cells that would otherwise be unable to initiate a metastatic niche. To test this hypothesis, we co-inoculated PC3-ML and AR(+) PCa cells and evaluated their metastatic abilities using two-color labeling in both multispectral microscopy and bioluminescence imaging. We found that the presence of AR(-), IL-1β-expressing cells allowed AR(+) PCa cells both to colonize the bone and persist in macroscopic mixed tumors in the long term. Our study thus conclusively confirms the existence of AR(-) PCa cells in skeletal metastases, describes a negative correlation between AR and IL-1β expression, and implicates this cytokine in a form of metastatic cooperation between phenotypically distinct cancer cells. These discoveries may be exploited therapeutically to alter the microenvironment of the metastatic niche, with the aim to impair establishment and progression of bone metastatic disease in PCa patients. Citation Format: Kristina S. Shahriari, Fei Shen, QingXin Liu, Danielle L. Jernigan, Ramanpreet Kaur, Alessandro Fatatis. Interleukin-1β secreted into the bone metastatic niche by androgen receptor-negative prostate cancer cells enables skeletal metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5065. doi:10.1158/1538-7445.AM2015-5065

Therapeutic approaches aimed to prevent breast cancer cells from spreading to distant sites are commonly neglected, based on the indication that dissemination from primary tumors can occur early. However, mounting evidence indicates that cancer cells recirculating from established bone lesions could further seed and colonize both skeleton and soft-tissue organs, thus precipitating metastatic dissemination and accelerating disease progression. We have previously shown that breast circulating tumor cells (CTCs) rely on the chemokine receptor CX3CR1 for lodging to the skeleton. Here, we show that this receptor is overexpressed by all four main subtypes of breast adenocarcinoma and also abundantly detected in bone metastatic lesions from breast cancer patients. We have recently synthesized JMS-17-2, a novel small molecule inhibitor of CX3CR1. We tested this compound in pre-clinical animal models of metastases in which human breast cancer cells, labeled with fluorescent and bioluminescent markers, are converted into CTCs by direct grafting in the arterial blood circulation. Pre-incubating cancer cells with JMS-17-2 or treating animals with this compound (10 mg/Kg, i.p.) were equally effective in dramatically decreasing the number of breast CTCs that lodged to the skeleton. These disseminated tumor cells (DTCs) were detected 24 hours after grafting and enumerated by multispectral fluorescence microscopy of sequential bone tissue sections spanning the entire width of tibiae and femora. Notably, when animals were examined at two weeks after grafting, JMS-17-2 completely prevented metastases, indicating that the reduction in number of DTCs directly translates to an impaired tumor growth in the long term. Finally, JMS-17-2 was administered to animals harboring a limited number of small metastases in bone and soft-tissues and monitored by bioluminescence imaging for three weeks before sacrifice. Animals treated with the CX3CR1 antagonist remained at an oligometastatic stage whereas control animals showed a sharp increase in the number of metastatic lesions and overall tumor burden. The work presented here should generate a conceptual shift in the treatment strategies for breast cancer patients, paving the way to approaches aimed to counteract the seeding of additional lesions from existing metastases. We also introduce JMS-17-2 as the first CX3CR1 antagonist and lead compound of a class of potentially new drugs with a novel mechanism of action that could be added to the arsenal of therapies currently used to treat advanced breast adenocarcinoma. Citation Format: Fei Shen, Yun Zhang, Danielle Jernigan, Jie Yan, Fernando Garcia, Olimpia Meucci, Joseph Salvino, Alessandro Fatatis. A small-molecule antagonist of CX3CR1 impairs homing and colonization of breast cancer cells in the skeleton. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4116. doi:10.1158/1538-7445.AM2015-4116

Advanced prostate cancer (PCa) frequently spreads to the skeleton and bone metastases are a major cause of morbidity and the main cause of death from this malignancy. The effective treatment of PCa disseminated to the skeleton remains an unmet clinical need. This lack of curative therapeutic approaches stems from our limited knowledge of the molecular interactions underpinning the colonization, survival and growth of cancer cells in the bone microenvironment. In this study, we focused on the autocrine and paracrine interactions involving different phenotypes of Disseminated Tumor Cells (DTCs) and the surrounding bone marrow stroma. These functional interactions are likely to play a crucial role in dictating the fate of DTCs and their disruption could interfere with the supportive role of the metastatic niche. By using comparative gene expression analysis we have demonstrated that IL-1β and CXCL6 determine the bone-metastatic behavior of human prostate cancer cells inoculated in the left cardiac ventricle of SCID mice. Since the tumor-producing cancer cells secrete both signaling molecules, we speculated that their roles include the establishment of cross talk with the surrounding cells of the bone stroma. The stromal cells would reciprocate with an increased or de novo production of trophic factors that promote metastatic progression. These local factors could also support DTC types that would normally be unable to initiate a metastatic niche, resulting in a form of cancer cell cooperation at distant sites. To test this hypothesis, we generated skeletal lesions in mice using Td-Tomato-labeled metastatic cells inoculated via the intracardiac route, followed by a second inoculation with GFP-labeled PCa cells lacking metastatic potential. We found that under these conditions the resulting skeletal lesions were composed of both cell types, indicating that metastasis-initiating cells could support subsequent colonization by cancer cells that normally fail to survive in the bone microenvironment. We then simultaneously inoculated animals with both cell types and discovered that the supportive influence of metastasis-initiating cells on non-metastatic cells is exerted immediately after homing to the skeleton. Further, we used Laser Capture Microdissection (LCM) to confirm high expression of IL-1β and CXCL6 in DTCs in metastatic lesions and then to identify signaling molecules and stromal cell types implicated in this novel cooperation mechanism in bone-metastatic lesions. These data may help identify multiple drug targets that, if clinically exploited, could lead to a substantial reduction in metastatic burden and concomitant increase in quality of life in the advanced prostate cancer patient population. Citation Format: Qingxin Liu, Kristina Shahriari, Marina Sazanovich, Danielle Jernigan, Alessandro Fatatis. Cancer cell cooperation in the bone metastatic niche. [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 3943. doi:10.1158/1538-7445.AM2013-3943

Skeletal metastasis is the major cause of morbidity and death in patients with advanced prostate cancer (PCa). The propensity of cancer cells to progress into macroscopic lesions at secondary sites relies on the successful adaptation of Disseminated Tumor Cells (DTCs) to the new microenvironment. This requirement explains the predilection of most tumors to produce clinical metastases in selected organs although they can seed malignant cells also in several other tissues. It seems plausible that the metastatic fate of DTCs is dictated by the pleiotropic effects of specific genes and their encoded products establishing functional interactions with the surrounding stroma. The main objective of this study was to identify these molecular mediators of cell survival and growth in the bone with the ultimate goal of identifying effective therapeutic targets to counteract PCa metastases. Using a pre-clinical animal model of experimental metastasis, we have recently shown that exogenous over-expression of PDGFRα in non-metastatic prostate phenotypes conferred bone-metastatic potential and a monoclonal antibody targeting the receptor reduced bone metastatic tumors by more than 70%. Here we report that comparative gene-expression analyses conducted with PCa cells showing different intrinsic metastatic potential, as well as prostate phenotypes that fail to increase their metastatic behavior upon PDGFRα expression, resulted in a bone-metastatic gene signature including three genes encoding for soluble, secreted proteins. This suggests that DTCs with aggressive metastatic behavior modify the bone microenvironment in their favor, not only coercing the stroma to support their own survival, but also allowing other malignant phenotypes, that would otherwise fail to thrive, to grow into macroscopic tumors. We are currently testing this cell-cooperation in metastasis as well as functionally validating our three-gene signature by stably silencing or over-expressing each of the three newly identified genes in bone-metastatic and non-metastatic PCa cells, respectively. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3392. doi:1538-7445.AM2012-3392

Because of constant improvements in early detection, the majority of breast cancer patients are diagnosed when still in a localized or regional stage and considered to be viable candidates for breast-conserving surgery or lumpectomy. As only 37% of breasts are found tumor-free following initial lumpectomy, between 22 and 59% of patients currently need re-intervention because positive or close margins are detected. In addition, re-excision or adjuvant therapies, such as local irradiation, are normally started several weeks or even months after primary surgery. During this period, the stroma at the site of tumor removal, which is characterized by altered angiogenesis, immune cells infiltration and activation of cancer-associated fibroblasts, may be selecting for cancer stem phenotypes, increasing metastatic potential and promoting the dissemination to specific secondary organs. This study aims to: 1) establish whether surgery on primary breast tumors leads to the emergence of cellular phenotypes with higher malignancy, associated with an increase in local growth, invasiveness and metastatic potential; 2) determine whether breast surgery alters the organ-tropism of residual cancer cells. For our experiments, BALB/c mice were inoculated in the mammary fat pad with 4T1 murine breast cancer cells and developed tumors that were either allowed to progress or surgically excised. Approximately 50% of animals in the surgery group presented with local recurrence, although all the excised tumors had positive margins. Local recurrences showed higher Ki-67 staining compared to their respective primary tumors and these data were in agreement with human samples of primary and recurrent breast tumors. Interestingly, the overall survival of mice in the control and surgery with recurrence groups were similar. Furthermore, macroscopic lung metastases were detected in the control and local recurrence groups, whereas mice without post-surgery recurrence failed to develop lung metastatic lesions. Finally, differences in the dissemination and growth of 4T1 cells to brain and bone between control, surgery plus recurrence and surgery without recurrence groups were investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1416. doi:1538-7445.AM2012-1416