Induction of N-Ras degradation by flunarizine-mediated autophagy.

Ras GTPases are powerful drivers for tumorigenesis, but directly targeting Ras for treating cancer remains challenging. The growth and transforming activity of the aggressive basal-like breast cancer (BLBC) are driven by N-Ras. To target N-Ras in BLBC, this study screened existing pharmacologically active compounds for the new ability to induce N-Ras degradation, which led to the identification of flunarizine (FLN), previously approved for treating migraine and epilepsy. The FLN-induced N-Ras degradation was not affected by a 26S-proteasome inhibitor. Rather, it was blocked by autophagy inhibitors. Furthermore, N-Ras can be seen co-localized with active autophagosomes upon FLN treatment, suggesting that FLN alters the autophagy pathway to degrade N-Ras. Importantly, FLN treatment recapitulated the effect of N-RAS silencing in vitro by selectively inhibiting the growth of BLBC cells, but not that of breast cancer cells of other subtypes. In addition, in vivo FLN inhibited tumor growth of a BLBC xenograft model. In conclusion, this proof-of-principle study presents evidence that the autophagy pathway can be coerced by small molecule inhibitors, such as FLN, to degrade Ras as a strategy to treat cancer. FLN has low toxicity and should be further investigated to enrich the toolbox of cancer therapeutics.

Systems biology-based drug repositioning identifies digoxin as a potential therapy for groups 3 and 4 medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Although outcomes have improved in recent decades, new treatments are still needed to improve survival and reduce treatment-related complications. The MB subtypes groups 3 and 4 represent a particular challenge due to their intragroup heterogeneity, which limits the options for "rational" targeted therapies. Here, we report a systems biology approach to drug repositioning that integrates a nonparametric, bootstrapping-based simulated annealing algorithm and a 3D drug functional network to characterize dysregulated driver signaling networks, thereby identifying potential drug candidates. From more than 1300 drug candidates studied, we identified five members of the cardiac glycoside family as potentially inhibiting the growth of groups 3 and 4 MB and subsequently confirmed this in vitro. Systemic in vivo treatment of orthotopic patient-derived xenograft (PDX) models of groups 3 and 4 MB with digoxin, a member of the cardiac glycoside family approved for the treatment of heart failure, prolonged animal survival at plasma concentrations known to be tolerated in humans. These results demonstrate the power of a systematic drug repositioning method in identifying a potential treatment for MB. Our strategy could potentially be used to accelerate the repositioning of treatments for other human cancers that lack clearly defined rational targets.

Targeting Brain-Adaptive Cancer Stem Cells Prohibits Brain Metastatic Colonization of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) exhibits more traits possessed by cancer stem cells (CSC) than other breast cancer subtypes and is more likely to develop brain metastases. TNBC patients usually have shorter survival time after diagnosis of brain metastasis, suggesting an innate ability of TNBC tumor cells in adapting to the brain. In this study, we establish novel animal models to investigate early tumor adaptation in brain metastases by introducing both patient-derived and cell line-derived CSC-enriched brain metastasis tumorsphere cells into mice. We discovered astrocyte-involved tumor activation of protocadherin 7 (PCDH7)-PLCß-Ca2+-CaMKII/S100A4 signaling as a mediator of brain metastatic tumor outgrowth. We further identified and evaluated the efficacy of a known drug, the selective PLC inhibitor edelfosine, in suppressing the PCDH7 signaling pathway to prohibit brain metastases in the animal models. The results of this study reveal a novel signaling pathway for brain metastases in TNBC and indicate a promising strategy of metastatic breast cancer prevention and treatment by targeting organ-adaptive cancer stem cells.Significance: These findings identify a compound to block adaptive signaling between cancer stem cells and brain astrocytes. Cancer Res; 78(8); 2052-64. ©2018 AACR.

In Vivo Visualization and Characterization of Epithelial-Mesenchymal Transition in Breast Tumors.

The activation of the epithelial-to-mesenchymal transition (EMT) program is a critical step in cancer progression and metastasis, but visualization of this process at the single-cell level, especially in vivo, remains challenging. We established an in vivo approach to track the fate of tumor cells based on a novel EMT-driven fluorescent color switching breast cancer mouse model and intravital two-photon laser scanning microscopy. Specifically, the MMTV-PyMT, Rosa26-RFP-GFP, and Fsp1-Cre triple transgenic mouse model was used to monitor the conversion of RFP-positive epithelial cells to GFP-positive mesenchymal cells in mammary tumors under the control of the Fsp1 (ATL1) promoter, a gate-keeper of EMT initiation. RFP-positive cells were isolated from the tumors, sorted, and transplanted into mammary fat pads of SCID mice to monitor EMT during breast tumor formation. We found that the conversion from RFP- to GFP-positive and spindle-shaped cells was a gradual process, and that GFP-positive cells preferentially localized close to blood vessels, independent of tumor size. Furthermore, cells undergoing EMT expressed high levels of the HGF receptor, c-Met, and treatment of RFP-positive cells with the c-Met inhibitor, cabozantinib, suppressed the RFP-to-GFP conversion in vitro Moreover, administration of cabozantinib to mice with palpable RFP-positive tumors resulted in a silent EMT phenotype whereby GFP-positive cells exhibited reduced motility, leading to suppressed tumor growth. In conclusion, our imaging technique provides a novel opportunity for visualizing tumor EMT at the single-cell level and may help to reveal the intricacies underlying tumor dynamics and treatment responses. Cancer Res; 76(8); 2094-104. ©2016 AACR.

A lab-on-a-chip system integrating tissue sample preparation and multiplex RT-qPCR for gene expression analysis in point-of-care hepatotoxicity assessment.

A truly practical lab-on-a-chip (LOC) system for point-of-care testing (POCT) hepatotoxicity assessment necessitates the embodiment of full-automation, ease-of-use and "sample-in-answer-out" diagnostic capabilities. To date, the reported microfluidic devices for POCT hepatotoxicity assessment remain rudimentary as they largely embody only semi-quantitative or single sample/gene detection capabilities. In this paper, we describe, for the first time, an integrated LOC system that is somewhat close to a practical POCT hepatotoxicity assessment device - it embodies both tissue sample preparation and multiplex real-time RT-PCR. It features semi-automation, is relatively easy to use, and has "sample-in-answer-out" capabilities for multiplex gene expression analysis. Our tissue sample preparation module incorporating both a microhomogenizer and surface-treated paramagnetic microbeads yielded high purity mRNA extracts, considerably better than manual means of extraction. A primer preloading surface treatment procedure and the single-loading inlet on our multiplex real-time RT-PCR module simplify off-chip handling procedures for ease-of-use. To demonstrate the efficacy of our LOC system for POCT hepatotoxicity assessment, we perform a preclinical animal study with the administration of cyclophosphamide, followed by gene expression analysis of two critical protein biomarkers for liver function tests, aspartate transaminase (AST) and alanine transaminase (ALT). Our experimental results depict normalized fold changes of 1.62 and 1.31 for AST and ALT, respectively, illustrating up-regulations in their expression levels and hence validating their selection as critical genes of interest. In short, we illustrate the feasibility of multiplex gene expression analysis in an integrated LOC system as a viable POCT means for hepatotoxicity assessment.

Epithelial derived CTGF promotes breast tumor progression via inducing EMT and collagen I fibers deposition.

Interactions among tumor cells, stromal cells, and extracellular matrix compositions are mediated through cytokines during tumor progression. Our analysis of 132 known cytokines and growth factors in published clinical breast cohorts and our 84 patient-derived xenograft models revealed that the elevated connective tissue growth factor (CTGF) in tumor epithelial cells significantly correlated with poor clinical prognosis and outcomes. CTGF was able to induce tumor cell epithelial-mesenchymal transition (EMT), and promote stroma deposition of collagen I fibers to stimulate tumor growth and metastasis. This process was mediated through CTGF-tumor necrosis factor receptor I (TNFR1)-IκB autocrine signaling. Drug treatments targeting CTGF, TNFR1, and IκB signaling each prohibited the EMT and tumor progression.

Electro-acupuncture up-regulates astrocytic MCT1 expression to improve neurological deficit in middle cerebral artery occlusion rats.

AIMS: Cerebral ischemia is one of the common diseases treated by electro-acupuncture (EA). Although the clinical efficacy has been widely affirmed, the mechanisms of action leading to the health benefits are not understood. In this study, the role of EA in modulating the lactate energy metabolism and lactate transportation was explored on the middle cerebral artery occlusion (MCAO) ischemic rat model. MAIN METHODS: Repeated EA treatments once daily for 7 days were applied to the MCAO rats and neurological function evaluation was performed. Brain tissues were harvested for lactate concentration examination, immunohistochemical staining, Western blot and qRT-PCR analyses for the expressions of lactate transporter (monocarboxylate transporter 1, MCT1) and glial fibrillary acidic protein (GFAP). KEY FINDINGS: The animal behavioral tests showed that the 7-day EA treatments significantly promoted the recovery of neurological deficits in the MCAO rats, which correlated with the enhanced lactate energy metabolism in the ischemic brain. In the cortical ischemic area of the MCAO rats, EA treatments led to the activation of astrocytes, and induced a further increase of lactate transporter (monocarboxylate transporter 1, MCT1) expression in astrocytes at both protein and mRNA levels. SIGNIFICANCE: Our results suggest that the EA treatments activated lactate metabolism in the resident astrocytes around the ischemic area and up-regulated the expression of MCT1 in these astrocytes which facilitated the transfer of intracellular lactate to extracellular domain to be utilized by injured neurons to improve the neurological deficit.

Transcriptional signaling pathways inversely regulated in Alzheimer's disease and glioblastoma multiform.

Convincing epidemiological data suggest an inverse association between cancer and neurodegeneration, including Alzheimer's disease (AD). Since both AD and cancer are characterized by abnormal, but opposing cellular behavior, i.e., increased cell death in AD while excessive cell growth occurs in cancer, this motivates us to initiate the study into unraveling the shared genes and cell signaling pathways linking AD and glioblastoma multiform (GBM). In this study, a comprehensive bioinformatics analysis on clinical microarray datasets of 1,091 GBM and 524 AD cohorts was performed. Significant genes and pathways were identified from the bioinformatics analyses - in particular ERK/MAPK signaling, up-regulated in GBM and Angiopoietin Signaling pathway, reciprocally up-regulated in AD - connecting GBM and AD (P < 0.001), were investigated in details for their roles in GBM growth in an AD environment. Our results showed that suppression of GBM growth in an AD background was mediated by the ERK-AKT-p21-cell cycle pathway and anti-angiogenesis pathway.

Novel modeling of cancer cell signaling pathways enables systematic drug repositioning for distinct breast cancer metastases.

A new type of signaling network element, called cancer signaling bridges (CSB), has been shown to have the potential for systematic and fast-tracked drug repositioning. On the basis of CSBs, we developed a computational model to derive specific downstream signaling pathways that reveal previously unknown target-disease connections and new mechanisms for specific cancer subtypes. The model enables us to reposition drugs based on available patient gene expression data. We applied this model to repurpose known or shelved drugs for brain, lung, and bone metastases of breast cancer with the hypothesis that cancer subtypes have their own specific signaling mechanisms. To test the hypothesis, we addressed specific CSBs for each metastasis that satisfy (i) CSB proteins are activated by the maximal number of enriched signaling pathways specific to a given metastasis, and (ii) CSB proteins are involved in the most differential expressed coding genes specific to each breast cancer metastasis. The identified signaling networks for the three types of breast cancer metastases contain 31, 15, and 18 proteins and are used to reposition 15, 9, and 2 drug candidates for the brain, lung, and bone metastases. We conducted both in vitro and in vivo preclinical experiments as well as analysis on patient tumor specimens to evaluate the targets and repositioned drugs. Of special note, we found that the Food and Drug Administration-approved drugs, sunitinib and dasatinib, prohibit brain metastases derived from breast cancer, addressing one particularly challenging aspect of this disease.

Differential effects of low- and high-dose GW2974, a dual epidermal growth factor receptor and HER2 kinase inhibitor, on glioblastoma multiforme invasion.

Aberrant expression of epidermal growth factor receptor (EGFR; ErbB1) and HER2 (ErbB2) tyrosine kinases frequently occurs in glioblastoma multiforme (GBM) patients and is considered to be associated with tumor malignancy and poor patient prognosis. In the present study, a dual EGFR and HER2 inhibitor (GW2974) was evaluated for its effects in GBM in vitro and in vivo. Results showed that low-concentration GW2974 inhibited GBM cell invasion, whereas a high concentration of the same compound counteracted this effect. Similar results were observed in an intracranial GBM xenograft model, in which, although both doses of GW2974 slowed tumor growth, no improvement in survival was observed in mice treated with high-dose GW2974, presumably because of the augmentation of tumor invasion. By protein microarray and Western blot analyses, the p38 mitogen-activated protein kinase (MAPK) pathway was found to be activated in GBM cells under high-concentration GW2974. Additionally, blockage of the p38 MAPK pathway reproduced the inhibitory effect of low-concentration GW2974 on cell invasion. These data suggest that the p38 MAPK pathway might contribute to the differential regulation of cell invasion by GW2974. Taken together, our results indicate that GW2974 exerts different effects in GBM depending on drug dosage. This offers a new perspective on the role of GW2974 in tumor progression, providing a potential strategy for GBM treatment based on precise prescription.

Identification of novel small-molecule inhibitors of glioblastoma cell growth and invasion by high-throughput screening.

Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor with a very poor prognosis. Current therapies for GBM remain palliative and advances made in decades have resulted in only a slight improvement in treatment outcome. Exploring new therapeutic agents for GBM treatment, therefore, is of prime importance. In the present study, we performed a high-throughput screening for GBM cell growth and invasion, with an attempt to identify novel potential anti-GBM agents. An annotated compound library (LOPAC1280) of 1,280 pharmacologically active compounds was screened and ten compounds were validated and identified as inhibitors of GBM cell growth and invasion. Four of them, i.e., 6-nitroso-1,2-benzopyrone, S-(p-azidophenacyl) glutathione, phenoxybenzamine hydrochloride, and SCH-28080 have not been implicated in GBM cell growth and invasion previously, suggesting that they may serve as novel potential therapeutic agents for GBM treatment. In conclusion, novel inhibitors of GBM cell growth and invasion were identified in the present study, which provides a basis for the development of therapies for GBM, and may shed light on the molecular mechanisms underlying GBM cell behavior.

Dehydroepiandrosterone indirectly inhibits human osteoclastic resorption via activating osteoblastic viability by the MAPK pathway.

BACKGROUND: Dehydroepiandrosterone (DHEA) is widely known for its beneficial effect on postmenopausal osteoporosis, although the underlying mechanisms remain mainly unclear. In this study, we tried to determine the activation of mitogen-activated protein kinase signal pathways during DHEA treatment and the indirect role of osteoblasts (OBs) on osteoclasts under the DHEA treatment of postmenopausal osteoporosis. METHODS: Primary human OBs and osteoclast-like cells were cultured and, we pretreated OBs with or without U0126 (a highly selective inhibitor of both MEK1 and MEK2). The OBs were treated with DHEA. We then tested the effects of DHEA on human osteoblastic viability, osteoprotegerin production and the expression of phosphor-ERK1/2 (extracellular signal-regulated kinase). In the presence or absence of OBs, the function of osteoclastic resorption upon DHEA treatment was calculated. RESULTS: DHEA promoted the human osteoblastic proliferation and inhibited the osteoblastic apoptosis within the concentration range of 10(-8) - 10(-6) mol/L (P < 0.05, P < 0.01, respectively). Within the effective concentration range, the expression of phosphor-ERK1/2 and osteoprotegerin was increased by DHEA and blocked by U0126. In the presence of OBs, DHEA could significantly decrease the number and the area of bone resorption lacuna (P < 0.05 and P < 0.01, respectively). Without OBs, however, the effects of DHEA on the bone resorption lacuna were almost completely abolished. CONCLUSIONS: DHEA could indirectly inhibit the human osteoclastic resorption through promoting the osteoblastic viability and osteoprotegerin production, which is mediated by mitogen-activated protein kinases signal pathway involving the phosphor-ERK1/2.

A novel method of transcriptional response analysis to facilitate drug repositioning for cancer therapy.

Little research has been done to address the huge opportunities that may exist to reposition existing approved or generic drugs for alternate uses in cancer therapy. In addition, there has been little work on strategies to reposition experimental cancer agents for testing in alternate settings that could shorten their clinical development time. Progress in each area has lagged, in part, because of the lack of systematic methods to define drug off-target effects (OTE) that might affect important cancer cell signaling pathways. In this study, we addressed this critical gap by developing an OTE-based method to repurpose drugs for cancer therapeutics, based on transcriptional responses made in cells before and after drug treatment. Specifically, we defined a new network component called cancer-signaling bridges (CSB) and integrated it with a Bayesian factor regression model (BFRM) to form a new hybrid method termed CSB-BFRM. Proof-of-concept studies were conducted in breast and prostate cancer cells and in promyelocytic leukemia cells. In each system, CSB-BFRM analysis could accurately predict clinical responses to more than 90% of drugs approved by the U.S. Food and Drug Administration and more than 75% of experimental clinical drugs that were tested. Mechanistic investigation of OTEs for several high-ranking drug-dose pairs suggested repositioning opportunities for cancer therapy, based on the ability to enforce retinoblastoma-dependent repression of important E2F-dependent cell-cycle genes. Together, our findings establish new methods to identify opportunities for drug repositioning or to elucidate the mechanisms of action of repositioned drugs.

The effect of mTOR inhibition alone or combined with MEK inhibitors on brain metastasis: an in vivo analysis in triple-negative breast cancer models.

mTOR inhibitor rapamycin and its analogs are lipophilic, demonstrate blood-brain barrier penetration, and have shown promising antitumor effects in several types of refractory tumors. We thus try to explore the therapeutic effects of mTOR inhibitors on brain metastasis models. We examined the effects of different dose of mTOR inhibitors (rapamycin, Temsirolimus-CCI-779) on cell invasion in two brain metastatic breast cancer cell lines (MDA-MB231-BR and CN34-BrM2). Antibody microarray and immunoblotting were applied to detect signaling pathways underlying the dose differential drug effects. The in vivo effects of single drug (CCI-779), and drug combination of CCI-779 with SL327 (a brain penetrant MEK inhibitor) to eliminate the unfavorable activation of MAPK pathway were evaluated in MDA-MB231-BR brain metastases xenograft mice. The two mTOR inhibitors, rapamycin and CCI-779, inhibited the invasion of brain metastatic cells only at a moderate concentration level, which was lost at higher concentrations secondary to activation of the MAPK signaling pathway. Pharmacological inhibition of ERK1/2 by PD98059 and SL327 restored the anti-invasion effects of mTOR inhibition in vitro. In vivo, a significant decrease was noted in the average number of micro and large metastatic lesions as well as the whole brain GFP expression in the CCI-779 1 mg/kg/day treated group compared with that in the vehicle group (P < 0.05). However, 10 mg/kg CCI-779 treatment did not show significant anti-metastasis effect on the animal model. High-dose CCI-779 eliciting the ERK MAPK activation in the brain metastatic lesion was corroborated. Combined with the brain penetrant MEK inhibitor SL327, high-dose CCI-779 significantly reduces the brain metastasis, and the combination treatment prohibited perivascular invasion of tumor cells and inhibits tumor angiogenesis in vivo. This study provides evidence on the potential value of CCI-779 as well as CCI-779 + SL327 in prohibiting breast cancer brain metastasis.

Aurora-B mediated ATM serine 1403 phosphorylation is required for mitotic ATM activation and the spindle checkpoint.

The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell-cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in shortened mitotic timing and a defective spindle checkpoint, and that abrogation of ATM Ser1403 phosphorylation leads to this spindle checkpoint defect. We also demonstrate that mitotically activated ATM phosphorylates Bub1, a critical kinetochore protein, on Ser314. ATM-mediated Bub1 Ser314 phosphorylation is required for Bub1 activity and is essential for the activation of the spindle checkpoint. Collectively, our data highlight mechanisms of a critical function of ATM in mitosis.

Real-time monitoring of cell viability using direct electrical measurement with a patch-clamp microchip.

Real-time tagless monitoring of cell viability using patch-clamp microchips is reported and validated by using fluorescence imaging techniques for the first time. Specifically, four human breast cancer cell lines (MDA-MB231, MDA-MB231-brain metastatic subline (abbreviated as MB231-BR), MB231-BR over-expressing HER2 gene (MB231-BR-HER2), and MB231-BR-vector control for the HER2 (MB231-BR-vector)) have been used for these studies. Systematic experiments on these cells found that the seal impedance/resistance of cells captured by the micro-pipettes always decreases during the process when the cell loses its viability, and therefore it is a valid indicator of live or dead cells. Systematic experiments also found that the Mega-seal of patch-clamp microchip is sufficient for monitoring cell viability. Given its simplicity of direct electrical measurement of cells without fluorescence labeling, this technology may provide an efficient technical platform to monitor the drug effects on cells, thereby significantly benefiting high throughput drug screening and discovery process.

Characterization of a human tumorsphere glioma orthotopic model using magnetic resonance imaging.

Magnetic resonance imaging (MRI) is the imaging modality of choice by which to monitor patient gliomas and treatment effects, and has been applied to murine models of glioma. However, a major obstacle to the development of effective glioma therapeutics has been that widely used animal models of glioma have not accurately recapitulated the morphological heterogeneity and invasive nature of this very lethal human cancer. This deficiency is being alleviated somewhat as more representative models are being developed, but there is still a clear need for relevant yet practical models that are well-characterized in terms of their MRI features. Hence we sought to chronicle the MRI profile of a recently developed, comparatively straightforward human tumor stem cell (hTSC) derived glioma model in mice using conventional MRI methods. This model reproduces the salient features of gliomas in humans, including florid neoangiogenesis and aggressive invasion of normal brain. Accordingly, the variable, invasive morphology of hTSC gliomas visualized on MRI duplicated that seen in patients, and it differed considerably from the widely used U87 glioma model that does not invade normal brain. After several weeks of tumor growth the hTSC model exhibited an MRI contrast enhancing phenotype having variable intensity and an irregular shape, which mimicked the heterogeneous appearance observed with human glioma patients. The MRI findings reported here support the use of the hTSC glioma xenograft model combined with MRI, as a test platform for assessing candidate therapeutics for glioma, and for developing novel MR methods.

Diffusion tensor-based fast marching for modeling human brain connectivity network.

Diffusion tensor imaging (DTI) is an effective modality in studying the connectivity of the brain. To eliminate possible biases caused by fiber extraction approaches due to low spatial resolution of DTI and the number of fibers obtained, the fast marching (FM) algorithm based on the whole diffusion tensor information is proposed to model and study the brain connectivity network. Our observation is that the connectivity extracted from the whole tensor field would be more robust and reliable for constructing brain connectivity network using DTI data. To construct the connectivity network, in this paper, the arrival time map and the velocity map generated by the FM algorithm are combined to define the connectivity strength among different brain regions. The conventional fiber tracking-based and the proposed tensor-based FM connectivity methods are compared, and the results indicate that the connectivity features obtained using the FM-based method agree better with the neuromorphical studies of the human brain.

Drug effects analysis on cells using a high throughput microfluidic chip.

Usually cell-based assay is performed using titer plates. Because of the large library of chemical compounds, robust and rapid methods are required to find, refine and test a potential drug candidate in an efficient manner. In this article, the drug effects analysis on human breast cancer cells with a droplet microfluidic chip is reported. Each droplet serves as a nanoliter-volume titer plate and contains a human breast cancer cell MDA-MB-231, Cytochalasin D drug solution and cell viability indicator such as Calcein AM, which emits cytoplasmic green fluorescence. The drug effects on each cell are monitored in real time using a fluorescence microscope and by analyzing the fluorescence image of each cell. Clear change of the cell shape and size has been observed after the drug treatment, which is similar to that of conventional petri dish technique, suggesting this approach is a potential viable technical platform for drug effect analysis and for high throughput drug screen and discovery.

Treating triple-negative breast cancer by a combination of rapamycin and cyclophosphamide: an in vivo bioluminescence imaging study.

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, has been shown to inhibit the growth of oestrogen positive breast cancer. However, triple-negative (TN) breast cancer is resistant to rapamycin treatment in vitro. We set to test a combination treatment of rapamycin with DNA-damage agent, cyclophosphamide, in a TN breast cancer model. By binding to and disrupting cellular DNA, cyclophosphamide kills cells via interfering with their normal functions. We assessed the responses of nude mice bearing tumour xenografts of TN MDA-MB-231 cells to the combination of rapamycin and cyclophosphamide in both orthotopic mammary and lung-metastasis models. We tracked tumour growth and metastasis by bioluminescent imaging and examined the expression of Ki67, CD34 and HIF-1alpha in tumour tissues by immunohistochemistry and apoptosis index with TUNEL assay, and found that MDA-MB-231 cells are sensitive to rapamycin therapy in orthotopic mammary, but not in lung with metastasis. Rapamycin when combined with cyclophosphamide is found to have a more significant effect in reducing tumour volume and metastasis with a much improved survival rate. Our data also show that the sensitivity of TN tumours to rapamycin is associated with the microenvironment of the tumour cells. The data indicate that in a relatively hypoxic environment HIF-1alpha may play a role in mediating the anti-cancer effect of rapamycin and cyclophosphamide may prevent the feedback activation of Akt by rapamycin. Overall our results show that rapamycin plus cyclophosphamide can achieve an improved efficacy in suppressing tumour growth and metastasis, suggesting that the combination therapy can be a promising treatment option for TN cancer.