Fibrinolytic System and Cancer: Diagnostic and Therapeutic Applications.

Fibrinolysis is a crucial physiological process that helps to maintain a hemostatic balance by counteracting excessive thrombosis. The components of the fibrinolytic system are well established and are associated with a wide array of physiological and pathophysiological processes. The aberrant expression of several components, especially urokinase-type plasminogen activator (uPA), its cognate receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), has shown a direct correlation with increased tumor growth, invasiveness, and metastasis. As a result, targeting the fibrinolytic system has been of great interest in the field of cancer biology. Even though there is a plethora of encouraging preclinical evidence on the potential therapeutic benefits of targeting the key oncogenic components of the fibrinolytic system, none of them made it from "bench to bedside" due to a limited number of clinical trials on them. This review summarizes our existing understanding of the various diagnostic and therapeutic strategies targeting the fibrinolytic system during cancer.

S-adenosylmethionine in combination with decitabine shows enhanced anti-cancer effects in repressing breast cancer growth and metastasis.

Abnormal DNA methylation orchestrates many of the cancer-related gene expression irregularities such as the inactivation of tumour suppressor genes through hypermethylation as well as activation of prometastatic genes through hypomethylation. The fact that DNA methylation abnormalities can be chemically reversed positions the DNA methylation machinery as an attractive target for anti-cancer drug development. However, although in vitro studies suggested that targeting concordantly hypo- and hypermethylation is of benefit in suppressing both oncogenic and prometastatic functions of breast cancer cells, this has never been tested in a therapeutic setting in vivo. In this context, we investigated the combined therapeutic effects of an approved nutraceutical agent S-adenosylmethionine (SAM) and FDA-approved hypomethylating agent decitabine using the MDA-MB-231 xenograft model of breast cancer and found a pronounced reduction in mammary tumour volume and lung metastasis compared to the animals in the control and monotherapy treatment arms. Immunohistochemical assessment of the primary breast tumours showed a significantly reduced expression of proliferation (Ki-67) and angiogenesis (CD31) markers following combination therapy as compared to the control group. Global transcriptome and methylome analyses have revealed that the combination therapy regulates genes from several key cancer-related pathways that are abnormally expressed in breast tumours. To our knowledge, this is the first preclinical study demonstrating the anti-cancer therapeutic potential of using a combination of methylating (SAM) and demethylating agent (decitabine) in vivo. Results from this study provide a molecularly founded rationale for clinically testing a combination of agents targeting the epigenome to reduce the morbidity and mortality from breast cancer.

DNA methylation signatures of Prostate Cancer in peripheral T-cells.

BACKGROUND: Prostate Cancer (PCa) is the second most common cancer in men where advancements have been made for early detection using imaging techniques, however these are limited by lesion size. Immune surveillance has emerged as an effective approach for early detection and to monitor disease progression. In recent studies, we have shown that host peripheral blood immune cells undergo changes in DNA methylation in liver and breast cancer. METHODS: In the current study, we examined the DNA methylation status of peripheral blood T cells of men with positive biopsy for PCa versus men with negative biopsy having benign prostate tissue, defined as controls. T cells DNA was isolated and subjected to Illumina Infinium methylation EPIC array and validated using Illumina amplicon sequencing and pyrosequencing platforms. RESULTS: Differential methylation of 449 CG sites between control and PCa T cell DNA showed a correlation with Gleason score (p < 0.05). Two hundred twenty-three differentially methylated CGs between control and PCa (Ƨ +/- 10%, p < 0.05), were enriched in pathways involved in immune surveillance system. Three CGs which were found differentially methylated following DMP (Differentially methylated probes) analysis of ChAMP remained significant after BH (Benjamini-Hochberg) correction, of which, 2 CGs were validated. Predictive ability of combination of these 3 CGs (polygenic methylation score, PMS) to detect PCa had high sensitivity, specificity and overall accuracy. PMS also showed strong positive correlation with Gleason score and tumor volume of PCa patients. CONCLUSIONS: Results from the current study provide for the first-time a potential role of DNA methylation changes in peripheral T cells in PCa. This non-invasive methodology may allow for early intervention and stratification of patients into different prognostic groups to reduce PCa associated morbidity from repeat invasive prostate biopsies and design therapeutic strategy to reduce PCa associated mortality.

Targeting DNA Hypomethylation in Malignancy by Epigenetic Therapies.

DNA methylation is a chemically reversible epigenetic modification that regulates the chromatin structure and gene expression, and thereby takes part in various cellular processes like embryogenesis, genomic imprinting, X-chromosome inactivation, and genome stability. Alterations in the normal methylation levels of DNA may contribute to the development of pathological conditions like cancer. Even though both hypo- and hypermethylation-mediated abnormalities are prevalent in the cancer genome, the field of cancer epigenetics has been more focused on targeting hypermethylation. As a result, DNA hypomethylation-mediated abnormalities remained relatively less explored, and currently, there are no approved drugs that can be clinically used to target hypomethylation. Understanding the precise role of DNA hypomethylation is not only crucial from a mechanistic point of view but also for the development of pharmacological agents that can reverse the hypomethylated state of the DNA. This chapter focuses on the causes and impact of DNA hypomethylation in the development of cancer and describes the possible ways to pharmacologically target it, especially by using a naturally occurring physiologic agent S-adenosylmethionine (SAM).

DNA methylation signatures of breast cancer in peripheral T-cells.

BACKGROUND: Immune surveillance acts as a defense mechanism in cancer, and its disruption is involved in cancer progression. DNA methylation reflects the phenotypic identity of cells and recent data suggested that DNA methylation profiles of T cells and peripheral blood mononuclear cells (PBMC) are altered in cancer progression. METHODS: We enrolled 19 females with stage 1 and 2, nine with stage 3 and 4 and 9 age matched healthy women. T cells were isolated from peripheral blood and extracted DNA was subjected to Illumina 450 K DNA methylation array analysis. Raw data was analyzed by BMIQ, ChAMP and ComBat followed by validation of identified genes by pyrosequencing. RESULTS: Analysis of data revealed ~ 10,000 sites that correlated with breast cancer progression and established a list of 89 CG sites that were highly correlated (p < 0.01, r > 0.7, r < - 0.7) with breast cancer progression. The vast majority of these sites were hypomethylated and enriched in genes with functions in the immune system. CONCLUSIONS: The study points to the possibility of using DNA methylation signatures as a noninvasive method for early detection of breast cancer and its progression which need to be tested in clinical studies.

Synergistic effects of combined DNA methyltransferase inhibition and MBD2 depletion on breast cancer cells; MBD2 depletion blocks 5-aza-2'-deoxycytidine-triggered invasiveness.

5-Aza-2'-deoxycytidine (5-azaCdR) not only inhibits growth of non-invasive breast cancer cells but also increases their invasiveness through induction of pro-metastatic genes. Methylated DNA binding protein 2 (MBD2) is involved in silencing methylated tumor suppressor genes as well as activation of pro-metastatic genes. In this study, we show that a combination of MBD2 depletion and DNA methyltransferases (DNMT) inhibition in breast cancer cells results in a combined effect in vitro and in vivo, enhancing tumor growth arrest on one hand, while inhibiting invasiveness triggered by 5-azaCdR on the other hand. The combined treatment of MBD2 depletion and 5-azaCdR suppresses and augments distinct gene networks that are induced by DNMT inhibition alone. These data point to a potential new approach in targeting the DNA methylation machinery by combination of MBD2 and DNMT inhibitors.

Methyl-CpG binding domain protein 2 (Mbd2) drives breast cancer progression through the modulation of epithelial-to-mesenchymal transition.

Methyl-CpG-binding domain protein 2 (Mbd2), a reader of DNA methylation, has been implicated in different types of malignancies, including breast cancer. However, the exact role of Mbd2 in various stages of breast cancer growth and progression in vivo has not been determined. To test whether Mbd2 plays a causal role in mammary tumor growth and metastasis, we performed genetic knockout (KO) of Mbd2 in MMTV-PyMT transgenic mice and compared mammary tumor progression kinetics between the wild-type (PyMT-Mbd2+/+) and KO (PyMT-Mbd2-/-) groups. Our results demonstrated that deletion of Mbd2 in PyMT mice impedes primary tumor growth and lung metastasis at the experimental endpoint (postnatal week 20). Transcriptomic and proteomic analyses of primary tumors revealed that Mbd2 deletion abrogates the expression of several key determinants involved in epithelial-to-mesenchymal transition, such as neural cadherin (N-cadherin) and osteopontin. Importantly, loss of the Mbd2 gene impairs the activation of the PI3K/AKT pathway, which is required for PyMT-mediated oncogenic transformation, growth, and survival of breast tumor cells. Taken together, the results of this study provide a rationale for further development of epigenetic therapies targeting Mbd2 to inhibit the progression of breast cancer.

Cyclin D1 cooperates with p21 to regulate TGFß-mediated breast cancer cell migration and tumor local invasion.

INTRODUCTION: Deregulation of the cell cycle machinery is often found in human cancers. Modulations in the cell cycle regulator function and expression result not only in proliferative advantages, but also lead to tumor progression and invasiveness of the cancer. In particular, cyclin D1 and p21 are often over-expressed in human cancers, correlating with high tumor grade, poor prognosis and increased metastasis. This prompted us to investigate the role of the cyclin D1/p21 signaling axis downstream of transforming growth factor beta (TGFß) in breast cancer progression. METHODS: Cyclins mRNA and protein expressions were assessed by quantitative real-time PCR and Western blot in triple negative breast cancer cell lines. Co-localization and interaction between cyclin D1 and p21 were performed by immunocytochemistry and co-immunoprecipitation, respectively. Cell migration was assessed by wound healing and quantitative time-lapse imaging assays. In addition, the effects of cyclin D1 on cellular structure and actin organization were examined by staining with F-actin marker phalloidin and mesenchymal intermediate filament vimentin. Finally, a mammary fat pad xenograft mouse model was used to assess mammary tumor growth and local invasion. RESULTS: We found TGFß to specifically up-regulate the expression of cyclin D1 in triple negative breast cancer cells. Induction of cyclin D1 is also required for TGFß-mediated cell migration. Suppression of cyclin D1 expression not only resulted in a rounded and epithelial-like phenotype, but also prevented TGFß-induced vimentin and F-actin co-localization at the cell edge as well as invadopodia formation. Furthermore, TGFß promoted the nuclear co-localization and physical interaction between cyclin D1 and p21. The co-expression of cyclin D1 and p21 proteins are required for the initial steps of tumor development, as double knockdown of these two molecules prevented primary tumor formation in a Xenograft mouse model. Moreover, the in vivo studies indicated that locally advanced features of the invasive tumors, including skeletal muscle, mammary fat pad and lymphovascular invasion, as well as ulcerated skin, were attenuated in the absence of cyclin D1 and p21. CONCLUSIONS: Thus, our findings highlight the cyclin D1/p21 signaling axis as a critical regulator of TGFß-mediated tumor growth initiation and local tumor cell invasion, both in vitro and in vivo.

Methyl-CpG binding domain 2 (Mbd2) is an epigenetic regulator of autism-risk genes and cognition.

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2-/-) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2-/- mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2-/- mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFß-mediated breast cancer cell migration and invasion.

INTRODUCTION: Tumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFß)-mediated breast cancer cell migration and invasion. METHODS: A mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFß and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFß transcriptional activity was measured by a TGFß/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFß downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFß/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive. RESULTS: We found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFß. p21 and p/CAF regulates TGFß-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFß/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients. CONCLUSIONS: Together these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.

Epigenomic effects of vitamin D in colorectal cancer.

Vitamin D regulates a plethora of physiological processes in the human body and has been proposed to exert several anticancer effects. Epigenetics plays an important role in regulating vitamin D actions. In this review, we highlight the recent advances in the understanding of different epigenetic factors such as lncRNAs, miRNAs, methylation and acetylation influenced by vitamin D and its downstream targets in colorectal cancer to find more potential therapeutic targets. We discuss how vitamin D exerts anticancer properties through interactions between the vitamin D receptor and genes (e.g., SLC30A10), the microenvironment, microbiota and other factors in colorectal cancer. Developing therapeutic approaches targeting the vitamin D signaling system will be aided by a better knowledge of the epigenetic impact of vitamin D.

Vitamin D regulates various physiological processes in the body and could have anticancer effects. These anticancer effects are the result of interactions between many factors such as genes, the environment around the tumors, bacteria in the intestine, etc. in colorectal cancer. Epigenetic factors, including a big network of different molecules in the body that could control our genes without changing DNA, also play a role in regulating vitamin D. This review summarizes the advances in the understanding of different epigenetic factors related to vitamin D and colorectal cancer.

Co-Targeting Luminal B Breast Cancer with S-Adenosylmethionine and Immune Checkpoint Inhibitor Reduces Primary Tumor Growth and Progression, and Metastasis to Lungs and Bone.

Breast cancer (BCa) is the most prevalent cancer in females and has a high rate of mortality, especially due to increased metastasis to skeletal and non-skeletal sites. Despite the marked clinical accomplishment of immune checkpoint inhibitor (CPI) therapy in patients with several cancers, it has had limited success in luminal subtypes of BCa. Accordingly, recent efforts have focused on combination therapy with CPI, including epigenetic modulators, to increase response rates of CPI in luminal BCa. We have previously shown that S-adenosylmethionine (SAM), the ubiquitous methyl donor, has strong anti-cancer effects in various cancers, including all subtypes of BCa. In the current study, we took a novel approach and examined the effect of CPI alone and in combination with SAM on tumor growth and metastasis in a syngeneic mouse model of luminal B BCa. We showed that SAM decreases cell proliferation, colony-formation (survival), and invasion of luminal B BCa cell lines (Eo771, R221A) in vitro. In in vivo studies, in Eo771 tumor-bearing mice, either SAM or anti-PD-1 antibody treatment alone significantly reduced tumor growth and progression, while the SAM+anti-PD-1 combination treatment had the highest anti-cancer efficacy of all groups. The SAM+anti-PD-1 combination reduced the percentage of animals with lung metastasis, as well as total metastatic lesion area, compared to control. Additionally, the SAM+anti-PD-1 combination significantly reduced the skeletal lesion area and protected tibial integrity to a greater extent than the monotherapies in an Eo771 bone metastasis model. Transcriptome analysis of Eo771 primary tumors revealed significant downregulation of pro-metastatic genes, including Matrix metalloproteinases (MMPs) and related pathways. On the other hand, CD8+ T cell infiltration, CD8+ T cell cytotoxicity (elevated granzymes), and immunostimulatory genes and pathways were significantly upregulated by the combination treatment. The results presented point to a combination of SAM with CPI as a possible treatment for luminal B BCa that should be tested in clinical studies.

Role of epigenetics in cancer initiation and progression.

The epigenome which comprises DNA methylation, histone modifications, chromatin structures and non-coding RNAs controls gene expression patterns. In cancer cells, there are aberrant changes in the epigenome. The question in cancer epigenetics is that whether these changes are the cause of cell transformation, or rather the consequence of it. We will discuss the epigenetic phenomenon in cancer, as well as the recent interests in the epigenetic reprogramming events, and their implications in the cancer stem cell theory. We will also look at the progression of cancers as they become more aggressive, with focus on the role of epigenetics in tumor metastases exemplified with the urokinase plasminogen activator (uPA) system. Last but not least, with therapeutics intervention in mind, we will highlight the importance of balance in the design of epigenetic based anti-cancer therapeutic strategies.

Role of Methylation in Pro- and Anti-Cancer Immunity.

DNA and RNA methylation play a vital role in the transcriptional regulation of various cell types including the differentiation and function of immune cells involved in pro- and anti-cancer immunity. Interactions of tumor and immune cells in the tumor microenvironment (TME) are complex. TME shapes the fate of tumors by modulating the dynamic DNA (and RNA) methylation patterns of these immune cells to alter their differentiation into pro-cancer (e.g., regulatory T cells) or anti-cancer (e.g., CD8+ T cells) cell types. This review considers the role of DNA and RNA methylation in myeloid and lymphoid cells in the activation, differentiation, and function that control the innate and adaptive immune responses in cancer and non-cancer contexts. Understanding the complex transcriptional regulation modulating differentiation and function of immune cells can help identify and validate therapeutic targets aimed at targeting DNA and RNA methylation to reduce cancer-associated morbidity and mortality.

An enhanced chemopreventive effect of methyl donor S-adenosylmethionine in combination with 25-hydroxyvitamin D in blocking mammary tumor growth and metastasis.

Therapeutic targeting of metastatic breast cancer still remains a challenge as the tumor cells are highly heterogenous and exploit multiple pathways for their growth and metastatic spread that cannot always be targeted by a single-agent monotherapy regimen. Therefore, a rational approach through simultaneous targeting of several pathways may provide a better anti-cancer therapeutic effect. We tested this hypothesis using a combination of two nutraceutical agents S-adenosylmethionine (SAM) and Vitamin D (Vit. D) prohormone [25-hydroxyvitamin D; '25(OH)D'] that are individually known to exert distinct changes in the expression of genes involved in tumor growth and metastasis. Our results show that both SAM and 25(OH)D monotherapy significantly reduced proliferation and clonogenic survival of a panel of breast cancer cell lines in vitro and inhibited tumor growth, lung metastasis, and breast tumor cell colonization to the skeleton in vivo. However, these effects were significantly more pronounced in the combination setting. RNA-Sequencing revealed that the transcriptomic footprint on key cancer-related signaling pathways is broader in the combination setting than any of the monotherapies. Furthermore, comparison of the differentially expressed genes from our transcriptome analyses with publicly available cancer-related dataset demonstrated that the combination treatment upregulates genes from immune-related pathways that are otherwise downregulated in bone metastasis in vivo. Since SAM and Vit. D are both approved nutraceuticals with known safety profiles, this combination treatment may serve as a novel strategy to reduce breast cancer-associated morbidity and mortality.

uPAR antibody (huATN-658) and Zometa reduce breast cancer growth and skeletal lesions.

Urokinase plasminogen activator receptor (uPAR) is implicated in tumor growth and metastasis due to its ability to activate latent growth factors, proteases, and different oncogenic signaling pathways upon binding to different ligands. Elevated uPAR expression is correlated with the increased aggressiveness of cancer cells, which led to its credentialing as an attractive diagnostic and therapeutic target in advanced solid cancer. Here, we examine the antitumor effects of a humanized anti-uPAR antibody (huATN-658) alone and in combination with the approved bisphosphonate Zometa (Zoledronic acid) on skeletal lesion through a series of studies in vitro and in vivo. Treatment with huATN-658 or Zometa alone significantly decreased human MDA-MB-231 cell proliferation and invasion in vitro, effects which were more pronounced when huATN-658 was combined with Zometa. In vivo studies demonstrated that huATN-658 treatment significantly reduced MDA-MB-231 primary tumor growth compared with controls. In a model of breast tumor-induced bone disease, huATN-658 and Zometa were equally effective in reducing skeletal lesions. The skeletal lesions were significantly reduced in animals receiving the combination of huATN-658 + Zometa compared with monotherapy treatment. These effects were due to a significant decrease in osteoclastic activity and tumor cell proliferation in the combination treatment group. Transcriptome analysis revealed that combination treatment significantly changes the expression of genes from signaling pathways implicated in tumor progression and bone remodeling. Results from these studies provide a rationale for the continued development of huATN-658 as a monotherapy and in combination with currently approved agents such as Zometa in patients with metastatic breast cancer.

SH2B1beta (SH2-Bbeta) enhances expression of a subset of nerve growth factor-regulated genes important for neuronal differentiation including genes encoding urokinase plasminogen activator receptor and matrix metalloproteinase 3/10.

Previous work showed that the adapter protein SH2B adapter protein 1beta (SH2B1) (SH2-B) binds to the activated form of the nerve growth factor (NGF) receptor TrkA and is critical for both NGF-dependent neurite outgrowth and maintenance. To identify SH2B1beta-regulated genes critical for neurite outgrowth, we performed microarray analysis of control PC12 cells and PC12 cells stably overexpressing SH2B1beta (PC12-SH2B1beta) or the dominant-negative SH2B1beta(R555E) [PC12-SH2B1beta(R555E)]. NGF-induced microarray expression of Plaur and Mmp10 genes was greatly enhanced in PC12-SH2B1beta cells, whereas NGF-induced Plaur and Mmp3 expression was substantially depressed in PC12-SH2B1beta(R555E) cells. Plaur, Mmp3, and Mmp10 are among the 12 genes most highly up-regulated after 6 h of NGF. Their protein products [urokinase plasminogen activator receptor (uPAR), matrix metalloproteinase 3 (MMP3), and MMP10] lie in the same pathway of extracellular matrix degradation; uPAR has been shown previously to be critical for NGF-induced neurite outgrowth. Quantitative real-time PCR analysis revealed SH2B1beta enhancement of NGF induction of all three genes and the suppression of NGF induction of all three when endogenous SH2B1 was reduced using short hairpin RNA against SH2B1 and in PC12-SH2B1beta(R555E) cells. NGF-induced levels of uPAR and MMP3/10 and neurite outgrowth through Matrigel (MMP3-dependent) were also increased in PC12-SH2B1beta cells. These results suggest that SH2B1beta stimulates NGF-induced neuronal differentiation at least in part by enhancing expression of a specific subset of NGF-sensitive genes, including Plaur, Mmp3, and/or Mmp10, required for neurite outgrowth.

A Src/Abl kinase inhibitor, SKI-606, blocks breast cancer invasion, growth, and metastasis in vitro and in vivo.

The central role of Src in the development of several malignancies, including breast cancer, and the accumulating evidence of its interaction with receptor tyrosine kinases, integrins, and steroid receptors have identified it as an attractive therapeutic target. In the current study, we have evaluated the effect of a Src/Abl kinase inhibitor, SKI-606, on breast cancer growth, migration, invasion, and metastasis. Treatment of human breast cancer cells MDA-MB-231 with SKI-606 caused a marked inhibition of cell proliferation, invasion, and migration by inhibiting mitogen-activated protein kinase and Akt phosphorylation. For in vivo studies, MDA-MB-231 cells transfected with the plasmid encoding green fluorescent protein (GFP; MDA-MB-231-GFP) were inoculated into the mammary fat pads of female BALB/c nu/nu mice. Once tumor volume reached 30 to 50 mm(3), animals were randomized and treated with vehicle alone or 150 mg/kg SKI-606 by daily oral gavage. Experimental animals receiving SKI-606 developed tumors of significantly smaller volume (45-54%) compared with control animals receiving vehicle alone. Analysis of lungs, liver, and spleen of these animals showed a significant decrease in GFP-positive tumor metastasis in animals receiving SKI-606 at a dose that was well tolerated. Western blot analysis and immunohistochemical analysis of primary tumors showed that these effects were due to the ability of SKI-606 to block tumor cell proliferation, angiogenesis, growth factor expression, and inhibition of Src-mediated signaling pathways in vivo. Together, the results from these studies provide compelling evidence for the role of Src inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.