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 (18)F-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.

Application of metabolic PET imaging in radiation oncology.

Positron emission tomography (PET) is a noninvasive imaging technique that provides functional or metabolic assessment of normal tissue or disease conditions and is playing an increasing role in cancer radiotherapy planning. (18)F-Fluorodeoxyglucose PET imaging (FDG-PET) is widely used in the clinic for tumor imaging due to increased glucose metabolism in most types of tumors; its role in radiotherapy management of various cancers is reviewed. In addition, other metabolic PET imaging agents at various stages of preclinical and clinical development are reviewed. These agents include radiolabeled amino acids such as methionine for detecting increased protein synthesis, radiolabeled choline for detecting increased membrane lipid synthesis, and radiolabeled acetate for detecting increased cytoplasmic lipid synthesis. The amino acid analogs choline and acetate are often more specific to tumor cells than FDG, so they may play an important role in differentiating cancers from benign conditions and in the diagnosis of cancers with either low FDG uptake or high background FDG uptake. PET imaging with FDG and other metabolic PET imaging agents is playing an increasing role in complementary radiotherapy planning.

Metabolic positron emission tomography imaging in cancer detection and therapy response.

Positron emission tomography (PET) is a noninvasive imaging technique that provides a functional or metabolic assessment of normal tissue or disease conditions. Fluorine 18-fluorodeoxyglucose PET imaging (FDG-PET) is widely used clinically for tumor imaging due to increased glucose metabolism in most types of tumors, and has been shown to improve the diagnosis and subsequent treatment of cancers. We review its use in cancer diagnosis, staging, restaging, and assessment of response to treatment. In addition, other metabolic PET imaging agents in pre-clinical research or clinical trial stages of development are discussed, including amino acid analogs based on increased protein synthesis, and choline, which is based on increased membrane lipid synthesis. Amino acid analogs and choline are more specific to tumor cells than FDG, so they play an important role in differentiating cancers from benign conditions and in the diagnosis of cancers with low FDG uptake or high background FDG uptake. For decades, researchers have shown that tumors display altered metabolic profiles with elevated uptake of glucose, amino acids, and lipids. This can be used for cancer diagnosis and monitoring of the therapeutic response with excellent signal-to-noise ratios.

Current molecular imaging positron emitting radiotracers in oncology.

Molecular imaging is one of the fastest growing areas of medical imaging. Positron emission tomography (PET) has been widely used in the clinical management of patients with cancer. Nuclear imaging provides biological information at the cellular, subcellular, and molecular level in living subjects with non-invasive procedures. In particular, PET imaging takes advantage of traditional diagnostic imaging techniques and introduces positron-emitting probes to determine the expression of indicative molecular targets at different stages of cancer. (18)F-fluorodeoxyglucose ((18)F-FDG), the only FDA approved oncological PET tracer, has been widely utilized in cancer diagnosis, staging, restaging, and even monitoring response to therapy; however, (18)F-FDG is not a tumor-specific PET tracer. Over the last decade, many promising tumor-specific PET tracers have been developed and evaluated in preclinical and clinical studies. This review provides an overview of the current non-(18)F-FDG PET tracers in oncology that have been developed based on tumor characteristics such as increased metabolism, hyperproliferation, angiogenesis, hypoxia, apoptosis, and tumor-specific antigens and surface receptors.

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

PURPOSE: L-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. METHODS: LAT1 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-[(18)F]fluorocyclobutane-1-carboxylic acid (anti-[(18)F]FACBC) PET was assessed for its diagnostic biomarker potential. RESULTS: Normal 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-[(18)F]FACBC, has been demonstrated to be an excellent PET tracer for the non-invasive imaging of malignant breast cancer using an orthotopic animal model. CONCLUSIONS: The overexpression of LAT1 is required for the progression of breast cancer. LAT1 represents a potential biomarker for therapy and diagnosis of breast cancer. Anti-[(18)F]FACBC that correlates with LAT1 function is a potential PET tracer for malignant breast tumor imaging.

Dipyrimidine amines: a novel class of chemokine receptor type 4 antagonists with high specificity.

The C-X-C chemokine receptor type 4 (CXCR4)/stromal cell derived factor-1 (SDF-1 or CXCL12) interaction and the resulting cell signaling cascade play a key role in metastasis and inflammation. On the basis of the previously published CXCR4 antagonist 5 (WZ811), a series of novel nonpeptidic anti-CXCR4 small molecules have been designed and synthesized to improve potency. Following a structure-activity profile around 5, more advanced compounds in the N,N'-(1, 4-phenylenebis(methylene)) dipyrimidin-2-amines series were discovered and shown to possess higher CXCR4 binding potential and specificity than 5. Compound 26 (508MCl) is the lead compound and exhibits subnanomolar potency in three in vitro assays including competitive binding, Matrigel invasion and Gα(i) cyclic adenosine monophosphate (cAMP) modulation signaling. Furthermore, compound 26 displays promising effects by interfering with CXCR4 function in three mouse models: paw inflammation, Matrigel plug angiogenesis, and uveal melanoma micrometastasis. These data demonstrate that dipyrimidine amines are unique CXCR4 antagonists with high potency and specificity.

Discovery of small molecule CXCR4 antagonists.

In light of a proposed molecular mechanism for the C-X-C chemokine receptor type 4 (CXCR4) antagonist 1 (AMD3100), a template with the general structure 2 was designed, and 15 was identified as a lead by means of an affinity binding assay against the ligand-mimicking CXCR4 antagonist 3 (TN14003). Following a structure-activity profile around 15, the design and synthesis of a series of novel small molecular CXCR4 antagonists led to the discovery of 32 (WZ811). The compound shows subnanomolar potency (EC50 = 0.3 nM) in an affinity binding assay. In addition, when subjected to in vitro functional evaluation, 32 efficiently inhibits CXCR4/stromal cell-derived factor-1 (SDF-1)-mediated modulation of cyclic adenosine monophophate (cAMP) levels (EC50 = 1.2 nM) and SDF-1 induced Matrigel invasion (EC50 = 5.2 nM). Molecular field topology analysis (MFTA), a 2D quantitative structure-activity relationship (QSAR) approach based on local molecular properties (Van der Waals radii (VdW), atomic charges, and local lipophilicity), applied to the 32 series suggests structural modifications to improve potency.

Blockade of invasion and metastasis of breast cancer cells via targeting CXCR4 with an artificial microRNA.

miRNAs have been shown to function as regulatory molecules and to play an important role in cancer progression. Very little is currently known about the increasing invasion and metastasis of breast cancer due to the loss of expressive levels of certain miRNAs in breast tumor cells. In order to determine whether the CXCR4/SDF-1 pathway is regulated by expression of miRNAs, we designed and synthesized pre-miRNA against CXCR4. This double-stranded miRNA gene was ligated with a miR-155-based Block-iT Pol II miR RNAi Expression Vector (Invitrogen). Expression levels of CXCR4 in CXCR4-miRNA-transfected breast tumor cells had significantly declined. These cells exhibited reduced migration and invasion in vitro. Furthermore, they formed fewer lung metastases in vivo compared to ctrl-miRNA-transfected cells. These data support the conclusion that miRNA against CXCR4 can serve as an alterative means of therapy to lower CXCR4 expression and to block the invasion and metastasis of breast cancer 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.

Artificial tumor model suitable for monitoring 31P and 13C NMR spectroscopic changes during chemotherapy-induced apoptosis in human glioma cells.

An artificial tumor method was developed to study cells inside the sensitive volume of an NMR spectrometer during growth and apoptosis. The tumor was composed of a 50:50 mixture of tightly packed porous-collagen and nonporous-polystyrene microspheres. The porous collagen served as a growth surface for the tumor cells, and the nonporous polystyrene served as a structural support to limit compression of the packed bed during perfusion. The microspheres were held between two porous polyethylene discs that were tightly sealed inside the NMR perfusion chamber. The new method was evaluated with two cell types: a mouse mammary tumor line (EMT6/SF) and a human glioma line (SF188). The results indicate that for both lines, approximately 10(9) metabolically active cells could be sustained for at least 1 week in the 12-cm(3) artificial tumor. Further, cells undergoing chemotherapy-induced apoptosis (which is known to cause detachment of cells from their surroundings) were retained in the artificial tumor. In preliminary 31P NMR studies, glioma cells treated with temozolomide (TMZ) exhibited reduced phosphocholine (PCh) levels relative to glycerophosphocholine (GPC) and diphosphodiester (DPDE) levels. They also exhibited sharply reduced oxygen consumption and TCA cycle 13C labeling, while they retained glycolytic activity. These metabolic changes are consistent with those that would be expected during mitochondrially-mediated apoptosis.