Signal transducer and activator of transcription (STAT) 3 regulates many cardinal features of cancer including cancer cell growth, apoptosis resistance, DNA damage response, metastasis, immune escape, tumor angiogenesis, the Warburg effect, and oncogene addiction and has been validated as a drug target for cancer therapy. Several strategies have been employed to identify agents that target Stat3 in breast cancer but none has yet entered into clinical use. We used a high-throughput fluorescence microscopy search strategy to identify compounds in a drug-repositioning library (Prestwick library) that block ligand-induced nuclear translocation of Stat3 and identified piperlongumine (PL), a natural product isolated from the fruit of the pepper Piper longum. Piperlongumine inhibited Stat3 nuclear translocation, inhibited ligand-induced and constitutive Stat3 phosphorylation, and modulated expression of multiple Stat3-regulated genes. Surface plasmon resonance assay revealed that piperlongumine directly inhibited binding of Stat3 to its phosphotyrosyl (pY) peptide ligand. Phosphoprotein antibody array analysis revealed that PL does not modulate kinases known to activate Stat3 such as JAKs, Src kinase family members, or RTKs. PL inhibited anchorage-independent and anchorage-dependent growth of multiple breast cancer cell lines having increased pStat3 or total Stat3, and induced apoptosis. PL also inhibited mammosphere formation by tumor cells from patient derived xenografts (PDX). PL’s anti-tumorigenic function was causally linked to its Stat3-inhibitory effect. PL was non-toxic in mice up to a dose of 30 mg/Kg/day for 14 days and caused regression of breast cancer cell line xenografts in nude mice. Thus, PL represents a promising new agent for rapid entry into the clinic for use in treating breast cancer, as well as other cancers in which Stat3 plays a role.

C-X-C chemokine receptor 7 (CXCR7) is a known promoter of tumor progression and metastasis; however, little is known about its role in colon cancer. The aim of the present study was to investigate the function of CXCR7 in human colon cancer cells. CXCR7 mRNA levels were examined in HT-29 and SW-480 human colon cancer cell lines using a quantitative polymerase chain reaction. CXCR7-knockdown was performed with small interfering RNA and lentiviral-mediated gene delivery. Immunofluorescence (IF) was conducted to examine CXCR7 expression and localization in colon cancer cells. Cell survival and migration were evaluated using MTT and migration assays, respectively. HT-29 cells expressed higher levels of CXCR7 mRNA and were therefore used in subsequent experiments. IF staining revealed that the CXCR7 protein was expressed on the cell membrane, and its expression decreased following CXCR7-short hairpin RNA lentiviral transfection. Lentiviral CXCR7-knockdown resulted in decreased cell survival and migration; however, MTT assays revealed that the lentiviral vector itself was cytotoxic. This cytotoxicity was indicated as the cell survival of the negative control group cells was significantly decreased compared with that of the blank control group cells (P<0.05). In conclusion, it is becoming increasingly evident that CXCR7 plays a role in colon cancer promotion, suggesting that CXCR7 is a promising biomarker for chemokine receptor-based drug development. Furthermore, the fact that CXCR7 is expressed on the membrane and not intracellularly makes it a prime target for drug-based intervention.

Recent data suggest the existence of a unique subset of breast cancer cells capable of initiating tumor growth and giving rise to all other cells characteristic of a given tumor. These cells have been termed “cancer stem cells” or “tumor-initiating cells”. Breast cancer stem cells appear to be resistant to chemo- and radiotherapies and may be responsible for recurrence and metastasis in breast cancer patients. Identifying the cellular signaling pathways responsible for breast cancer stem cell maintenance and self-renewal represents a critical hurdle for developing effective therapeutics. The Stat3 pathway is a critical regulator of the function of normal stem cells, and shows altered expression in human breast cancers [4]. Moreover, IL-6, the Stat3 signaling agonist, is required for breast cancer stem cell function in human breast cancer cell lines. All these suggest an important role of Stat3 signaling in breast cancer stem cells. However, due to lack of method for pathway-activity-based live cell separation, whether Stat3 functions in the cancer stem cells themselves or whether it may function in surrounding niche cells remain unknown. We have constructed a lentiviral fluorescent reporter for Stat3 signaling which contains four copies of M67 Stat3 binding sites upstream of enhanced Green Fluorescent Protein. This reporter system enables FACS-sorting of cells with active Stat3 signaling and in vivo/in situ localization of Stat3 responsive cells. In addition, C188-9, the first Stat3 specific competitive inhibitor, has been developed in our collaborator's lab, which provides us an advanced tool in studying Stat3 function. We hypothesize that Stat3 signaling is preferentially active in stemlike subpopulation and depend on non-stem cancer cells to maintain its activation. Three aims to test this hypothesis include: 1. To test whether cancer cells with activated Stat3 signaling are enriched for breast cancer stem cells or whether they may serve as niche cells. 2. To test whether small molecule antagonists of Stat3 signaling can inhibit cancer stem cell function. 3. To identify novel targets of Stat3 signaling that may serve as indicators of responsiveness to Stat3 antagonist or predict treatment response. So far, our GFP reporters for Stat3 signaling effectively report Stat3 activity in both patient xenografts and human breast cancer cell lines in vivo and in vitro, which enables effective separation of Stat3+/Stat3- cells for functional Study. MDA231 and SUM159 tumors with reporters have been established. Tumor cells will be sorted according to Stat3 activity and perform limiting dilution and MSFE assays. Tumors with reporters will also be treated by C188-9, Chemo, or in combination to study synergistic effect of Stat3 inhibitors on tumor growth. Preliminary data based on inhibitor and limiting dilution studies suggested that Stat3 signaling is required for stem cell function. However, neither Stat3+ nor Stat3- cells are self-sufficient in performing these functions, indicating communications between subpopulations. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD08-05.