Zhongxing Liang

Inhibition of Breast Cancer Metastasis by Selective Synthetic Polypeptide against CXCR4

Metastasis shares many similarities with leukocyte trafficking. Among those chemokine receptors thought to be involved in hemopoietic cell homing, stromal cell-derived factor-1 and its receptor CXC chemokine receptor-4 (CXCR4) have received considerable attention. Like hemopoietic cell homing, levels of stromal cell-derived factor-1 are high at sites of breast cancer metastasis including lymph node, lung, liver, and the marrow. Moreover, CXCR4 expression is low in normal breast tissues and high in malignant tumors, suggesting that a blockade of CXCR4 might limit tumor metastasis. We therefore investigated the role of a synthetic antagonist 14-mer peptide (TN14003) in inhibiting metastasis in an animal model. Not only was TN14003 effective in limiting metastasis of breast cancer by inhibiting migration, but it may also prove useful as a diagnostic tool to identify CXCR4 receptor-positive tumor cells in culture and tumors in paraffin-embedded clinical samples.

Involvement of miR-326 in chemotherapy resistance of breast cancer through modulating expression of multidrug resistance-associated protein 1

Multidrug resistance-associated protein (MRP-1/ABCC1) transports a wide range of therapeutic agents and may play a critical role in the development of multidrug resistance (MDR) in tumor cells. However, the regulation of MRP-1 remains controversial. To explore whether miRNAs are involved in the regulation of MRP-1 expression and modulate the sensitivity of tumor cells to chemotherapeutic agents, we analyzed miRNA expression levels in VP-16-resistant MDR cell line, MCF-7/VP, in comparison with its parent cell line, MCF-7, using a miRNA microarray. MCF-7/VP overexpressed MRP-1 mRNA and protein not MDR-1 and BCRP. miR-326 was downregulated in MCF-7/VP compared to MCF-7. Additionally, miR-326 was downregulated in a panel of advanced breast cancer tissues and consistent reversely with expression levels of MRP-1. Furthermore, the elevated levels of miR-326 in the mimics-transfected VP-16-resistant cell line, MCF-7/VP, downregulated MRP-1 expression and sensitized these cells to VP-16 and doxorubicin. These findings demonstrate for the first time the involvement of miRNAs in multidrug resistance mediated by MRP-1 and suggest that miR-326 may be an efficient agent for preventing and reversing MDR in tumor cells.

CXC Chemokine Receptor-4 Antagonist Blocks Both Growth of Primary Tumor and Metastasis of Head and Neck Cancer in Xenograft Mouse Models

Squamous cell carcinoma of the head and neck (SCCHN) metastasizes to the lymph nodes and lungs. We have generated previously an orthotopic mouse model for head and neck metastasis and did in vivo selection of SCCHN cells through four rounds of serial metastases. A subpopulation of 686LN cells with high metastatic potential (686LN-Ms) was isolated. When the highly metastatic cells were compared with their low metastatic parental cells (686LN-Ps), we found that CXC chemokine receptor-4 (CXCR4) mRNA levels were significantly higher in the 686LN-Ms cells than the 686LN-Ps cells. Interestingly, the metastatic subclones had lost epithelial morphology and acquired mesenchymal features, which were maintained during cell expansion in vitro. This was featured by decreased E-cadherin and involucrin and increased vimentin and integrin beta(1). These results imply that CXCR4 and epithelial-mesenchymal transition markers can be potential biomarkers to identify the subpopulation of cells with high metastatic potential. Using the orthotopic SCCHN animal model, we showed that anti-CXCR4 treatment suppressed primary tumor growth by inhibiting tumor angiogenesis and prevented lung metastasis. Because the reduction of metastasis seen in the treated group could have resulted from 2-fold reduction in primary tumor size compared with that in the control group, we examined the effects of the CXCR4 antagonist in an experimental metastatic animal model in which 686LN-Ms cells were i.v. injected. 686LN-Ms cells failed to metastasize in the CXCR4 antagonist-treated group, whereas they metastasized to the lungs in the control group. Our data indicate that CXCR4 is an important target to inhibit tumor progression in SCCHN.

Regulation of miR-19 to Breast Cancer Chemoresistance Through Targeting PTEN

ABSTRACTPurposeTo explore whether miR-19 is involved in the regulation of multidrug resistance (MDR), one of the main causes of breast cancer mortality, and modulates sensitivity of tumor cells to chemotherapeutic agents.MethodsWe analyzed miRNA expression levels in three MDR cell lines in comparison with their parent cell line, MCF-7, using a miRNA microarray. We investigated whether inhibitor of miR-19 sensitized MDR cells to chemotherapeutic agents in vitro and in vivo.ResultsMiR-19 was overexpressed in all three MDR cell lines compared to their parental cell line, MCF-7. Expression levels of miR-19 in MDR cells were inversely consistent with those of PTEN. Inhibitor of miR-19a restored sensitivity of MDR cells to cytotoxic agents; administration of LNA-antimiR-19a, a chemo-modified miR-19a inhibitor, sensitized MDR cells to chemotherapeutic agents in vivo.ConclusionOur findings demonstrate, for the first time, involvement of miR-19 in multidrug resistance through modulation of PTEN and suggest that miR-19 may be a potential target for preventing and reversing MDR in tumor cells.

Development of a unique small molecule modulator of CXCR4.

Background Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. Methodology/Principal Findings We describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. Conclusions/Significance We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.

Antitumor Effect of 2-Methoxyestradiol in a Rat Orthotopic Brain Tumor Model

Grade 4 malignant glioma (GBM) is a fatal disease despite aggressive surgical and adjuvant therapies. The hallmark of GBM tumors is the presence of pseudopalisading necrosis and microvascular proliferation. These tumor cells are hypoxic and express hypoxia-inducible factor-1 (HIF-1), a prosurvival transcription factor that promotes formation of neovasculature through activation of target genes, such as vascular endothelial growth factor. Here, we evaluated whether 2-methoxyestradiol, a microtubule and HIF-1 inhibitor, would have therapeutic potential for this disease in a 9L rat orthotopic gliosarcoma model using a combination of noninvasive imaging methods: magnetic resonance imaging to measure the tumor volume and bioluminescence imaging for HIF-1 activity. After imaging, histologic data were subsequently evaluated to elucidate the drug action mechanism in vivo. Treatment with 2-methoxyestradiol (60-600 mg/kg/d) resulted in a dose-dependent inhibition of tumor growth. This effect was also associated with improved tumor oxygenation as assessed by pimonidazole staining, decreased HIF-1alpha protein levels, and microtubule destabilization as assessed by deacetylation. Our results indicate that 2-methoxyestradiol may be a promising chemotherapeutic agent for the treatment of malignant gliomas, with significant growth inhibition. Further studies are needed to assess the effect of low or intermediate doses of 2-methoxyestradiol in combination with chemotherapeutic agents in clinical studies focused on malignant gliomas. In addition to showing tumor growth inhibition, we identified three potential surrogate biomarkers to determine the efficacy of 2-methoxyestradiol therapy: decreased HIF-1alpha levels, alpha-tubulin acetylation, and degree of hypoxia as determined by pimonidazole staining.

MicroRNA-302 Replacement Therapy Sensitizes Breast Cancer Cells to Ionizing Radiation

ABSTRACTPurposeSolid tumors can be resistant or develop resistance to radiotherapy. The purpose of this study is to explore whether microRNA-302 is involved in radioresistance and can be exploited as a sensitizer to enhance sensitivity of breast cancer cells to radiation therapy.MethodsMiR-302 expression levels in radioresistant cell lines were analyzed in comparison with their parent cell lines. Furthermore, we investigated whether enforced expression of miR-302 sensitized radioresistant breast cancer cells to ionizing radiation in vitro and in vivo.ResultsMiR-302 was downregulated in irradiated breast cancer cells. Additionally, the expression levels of miR-302a were inversely correlated with those of AKT1 and RAD52, two critical regulators of radioresistance. More promisingly, miR-302a sensitized radioresistant breast cancer cells to radiation therapy in vitro and in vivo and reduced the expression of AKT1 and RAD52.ConclusionOur findings demonstrated that decreased expression of miR-302 confers radioresistance and restoration of miR-302 baseline expression sensitizes breast cancer cells to radiotherapy. These data suggest that miR-302 is a potential sensitizer to radiotherapy.

Potential Biomarker of L-type Amino Acid Transporter 1 in Breast Cancer Progression

PurposeL-type amino acid transporter 1 (LAT1) is essential for the transport of large neutral amino acids. However, its role in breast cancer growth remains largely unknown. The purpose of the study is to investigate whether LAT1 is a potential biomarker for the diagnosis and treatment of breast cancer.MethodsLAT1 mRNA and protein levels in breast cancer cell lines and tissues were analyzed. In addition, the effects of targeting LAT1 for the inhibition of breast cancer cell tumorigenesis were assessed with soft agar assay. The imaging of xenograft with anti-1-amino-3-[18F]fluorocyclobutane-1-carboxylic acid (anti-[18F]FACBC) PET was assessed for its diagnostic biomarker potential.ResultsNormal breast tissue or low malignant cell lines expressed low levels of LAT1 mRNA and protein, while highly malignant cancer cell lines and high-grade breast cancer tissue expressed high levels of LAT1. In addition, higher expression levels of LAT1 in breast cancer tissues were consistent with advanced-stage breast cancer. Furthermore, the blockade of LAT1 with its inhibitor, 2-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (BCH), or the knockdown of LAT1 with siRNA, inhibited proliferation and tumorigenesis of breast cancer cells. A leucine analog, anti-[18F]FACBC, has been demonstrated to be an excellent PET tracer for the non-invasive imaging of malignant breast cancer using an orthotopic animal model.ConclusionsThe overexpression of LAT1 is required for the progression of breast cancer. LAT1 represents a potential biomarker for therapy and diagnosis of breast cancer. Anti-[18F]FACBC that correlates with LAT1 function is a potential PET tracer for malignant breast tumor imaging.

Inhibition of breast cancer metastasis with microRNA-302a by downregulation of CXCR4 expression

Abstract Metastasis remains a main cause of mortality from breast cancer and an unresolved issue. The purpose of this study is to investigate the role of miR-302a in the development of breast cancer metastasis mediated by CXCR4, a critical regulator of metastasis, and to identify miR-302a as an effective therapeutic agent for therapy and prevention of breast cancer metastasis. Our studies show that miR-302a expression levels were downregulated in metastatic breast cancer cells and tumor tissues. Additionally, the expression levels of miR-302a were inversely correlated with CXCR4 levels. More promisingly, miR-302a inhibited the invasion and metastasis of breast cancer cells in vitro and in vivo and reduced the expression of CXCR4. Our findings demonstrated that the repression of miR-302a levels contributes to breast cancer metastasis and restoration of miR-302a baseline expression inhibits the invasion and metastasis of breast cancer cells. These data suggest that miR-302a mimics are potential therapeutic agents for breast cancer metastasis.

Whole-brain spectroscopic MRI biomarkers identify infiltrating margins in glioblastoma patients

BACKGROUND The standard of care for glioblastoma (GBM) is maximal safe resection followed by radiation therapy with chemotherapy. Currently, contrast-enhanced MRI is used to define primary treatment volumes for surgery and radiation therapy. However, enhancement does not identify the tumor entirely, resulting in limited local control. Proton spectroscopic MRI (sMRI), a method reporting endogenous metabolism, may better define the tumor margin. Here, we develop a whole-brain sMRI pipeline and validate sMRI metrics with quantitative measures of tumor infiltration. METHODS Whole-brain sMRI metabolite maps were coregistered with surgical planning MRI and imported into a neuronavigation system to guide tissue sampling in GBM patients receiving 5-aminolevulinic acid fluorescence-guided surgery. Samples were collected from regions with metabolic abnormalities in a biopsy-like fashion before bulk resection. Tissue fluorescence was measured ex vivo using a hand-held spectrometer. Tissue samples were immunostained for Sox2 and analyzed to quantify the density of staining cells using a novel digital pathology image analysis tool. Correlations among sMRI markers, Sox2 density, and ex vivo fluorescence were evaluated. RESULTS Spectroscopic MRI biomarkers exhibit significant correlations with Sox2-positive cell density and ex vivo fluorescence. The choline to N-acetylaspartate ratio showed significant associations with each quantitative marker (Pearsons ρ = 0.82, P < .001 and ρ = 0.36, P < .0001, respectively). Clinically, sMRI metabolic abnormalities predated contrast enhancement at sites of tumor recurrence and exhibited an inverse relationship with progression-free survival. CONCLUSIONS As it identifies tumor infiltration and regions at high risk for recurrence, sMRI could complement conventional MRI to improve local control in GBM patients.