Most cases of breast cancer mortality are due to vascular metastasis. Breast cancer cells must intravasate through endothelial cells (ECs) to enter a blood vessel in the primary tumor and then adhere to ECs and extravasate at the metastatic site. In this study we demonstrate that inhibition of hypoxia-inducible factor activity (HIF) in breast cancer cells by RNA interference or digoxin treatment inhibits primary tumor growth and also inhibits the metastasis of breast cancer cells to the lungs by blocking the expression of angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM). ANGPTL4 is a secreted factor that inhibits EC-EC interaction, whereas L1CAM increases the adherence of breast cancer cells to ECs. Interference with HIF, ANGPTL4, or L1CAM expression inhibits vascular metastasis of breast cancer cells to the lungs.

No abstract

Purpose Glutamine addiction in c-MYC–overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved. Experimental Design A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress–mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. Results We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress–mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc–overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy. Conclusions AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA’s effectiveness provide a strong rationale for further clinical development, particularly for c-MYC–overexpressing breast cancers.

The factors that influence nanoparticle fate in vivo following systemic delivery remain an area of intense interest. Of particular interest is whether labeling with a cancer-specific antibody ligand ("active targeting") is superior to its unlabeled counterpart ("passive targeting"). Using models of breast cancer in three immune variants of mice, we demonstrate that intratumor retention of antibody-labeled nanoparticles was determined by tumor-associated dendritic cells, neutrophils, monocytes, and macrophages and not by antibody-antigen interactions. Systemic exposure to either nanoparticle type induced an immune response leading to CD8 + T cell infiltration and tumor growth delay that was independent of antibody therapeutic activity. These results suggest that antitumor immune responses can be induced by systemic exposure to nanoparticles without requiring a therapeutic payload. We conclude that immune status of the host and microenvironment of solid tumors are critical variables for studies in cancer nanomedicine and that nanoparticle technology may harbor potential for cancer immunotherapy.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal human malignancy with historically limited success in treatment. The role of aberrant Notch signaling, which requires the constitutive activation of γ-secretase, in the initiation and progression of PDA is well defined and inhibitors of this pathway are currently in clinical trials. Here we investigated the in vivo therapeutic effect of PF-03084014, a selective γ-secretase inhibitor, alone and in combination with gemcitabine in pancreatic cancer xenografts. PF-03084014 treatment inhibited the cleavage of nuclear Notch 1 intracellular domain and Notch targets Hes-1 and Hey-1. Gemcitabine treatment showed good response but not capable of inducing tumor regressions and targeting the tumor-resident cancer stem cells (CD24+CD44+ and ALDH+ tumor cells). A combination of PF-03084014 and gemcitabine treatment resulted tumor regression in 3 of 4 subcutaneously implanted xenograft models. PF-03084014, and in combination with gemcitabine reduced putative cancer stem cells, indicating that PF-03084014 target the especially dangerous and resilient cancer stem cells within pancreatic tumors. Tumor re-growth curves plotted after drug treatments demonstrated that the effect of the combination therapy was sustainable than that of gemcitabine. Notably, in a highly aggressive orthotopic model, PF-03084014 and gemcitabine combination was effective in inducing apoptosis, inhibition of tumor cell proliferation and angiogenesis, resulting in the attenuation of primary tumor growth as well as controlling metastatic dissemination, compared to gemcitabine treatment. In summary, our preclinical data suggest that PF-03084014 has greater anti-tumor activity in combination with gemcitabine in PDA and provides rationale for the further investigation of this combination in PDA.

Why breast cancers become resistant to tamoxifen despite continued expression of the estrogen receptor alpha (ERα) and what factors are responsible for high HER2 expression in these tumors remains an enigma. HOXB7 ChIP analysis followed by validation showed that HOXB7 physically interacts with ERα, and that the HOXB7-ERα complex enhances transcription of many ERα target genes including HER2. Investigating strategies for controlling HOXB7, our studies revealed that MYC, stabilized via phosphorylation mediated by EGFR-HER2 signaling, inhibits transcription of miRNA-196a, a HOXB7 repressor. This leads to increased expression of HOXB7, ER-target genes and HER2. Repressing MYC using small molecule inhibitors reverses these events, and causes regression of breast cancer xenografts. The MYC-HOXB7-HER2 signaling pathway is eminently targetable in endocrine-resistant breast cancer.

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1−/− mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK’s effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.

Development of drug resistance is a major factor limiting the continued success of cancer chemotherapy. To overcome drug resistance, understanding the underlying mechanism(s) is essential. We found that HOXC10 is over-expressed in primary carcinomas of the breast, and even more significantly in distant metastasis arising after failed chemotherapy. High HOXC10 expression correlates with shorter recurrence-free and overall survival in patients with estrogen receptor (ER)-negative breast cancer undergoing chemotherapy. We found that HOXC10 promotes survival in cells treated with doxorubicin, paclitaxel or carboplatin by suppressing apoptosis and upregulating NF-κB. Overexpressed HOXC10 increases S-phase specific DNA damage repair by homologous recombination (HR) and checkpoint recovery in cells at three important phases. For double strand break repair, HOXC10 recruits HR proteins at sites of DNA damage. It enhances resection and lastly, it resolves stalled replication forks, leading to initiation of DNA replication following DNA damage. We show that HOXC10 facilitates, but is not directly involved in DNA damage repair mediated by HR. HOXC10 achieves integration of these functions by binding to, and activating cyclin dependent kinase, CDK7, which regulates transcription by phosphorylating the carboxy-terminal domain of RNA polymerase II. Consistent with these findings, inhibitors of CDK7 reverse HOXC10-mediated drug resistance in cultured cells. Blocking HOXC10 function, therefore, presents a promising new strategy to overcome chemotherapy resistance in breast cancer.