STAT3 Inhibition as a Therapeutic Strategy for Chordoma.

Objective Signal transducer and activator of transcription (STAT) proteins regulate key cellular fate decisions including proliferation and apoptosis. STAT3 overexpression induces tumor growth in multiple neoplasms. STAT3 is constitutively activated in chordoma, a tumor with a high recurrence rate despite maximal surgical and radiation treatment. We hypothesized that a novel small molecule inhibitor of STAT3 (FLLL32) would induce significant cytotoxicity in sacral and clival chordoma cells. Methods Sacral (UCh1) and clival (UM-CHOR-1) chordoma cell lines were grown in culture (the latter derived from primary tumor explants). FLLL32 dosing parameters were optimized using cell viability assays. Antitumor potential of FLLL32 was assessed using clonal proliferation assays. Potential mechanisms underlying observed cytotoxicity were examined using immunofluorescence assays. Results FLLL32 induced significant cytotoxicity in UCh1 and UM-CHOR-1 chordoma cells, essentially eliminating all viable cells, correlating with observed downregulation in activated, phosphorylated STAT3 upon administration of FLLL32. Mechanisms underlying the observed cytotoxicity included increased apoptosis and reduced cellular proliferation through inhibition of mitosis. Conclusion As a monotherapy, FLLL32 induces potent tumor kill in vitro in chordoma cell lines derived from skull base and sacrum. This effect is mediated through inhibition of STAT3 phosphorylation, increased susceptibility to apoptosis, and suppression of cell proliferation.

Tenascin-C: a novel candidate marker for cancer stem cells in glioblastoma identified by tissue microarrays.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, with dismal survival outcomes. Recently, cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here, we have investigated the expression of TNC in tissue microarrays including 17 GBMs, 18 WHO grade III astrocytomas, 15 WHO grade II astrocytomas, 4 WHO grade I astrocytomas, and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas, whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133(+)/TNC(+) and CD133(-)/TNC(+) cell populations is much higher than that of the CD133(+)/TNC(-) and CD133(-)/TNC(-) populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs, where the TNC(+) population is identified as a CSC population overlapping with part of the CD133(-) cell population.

KIT expression and methylation in medulloblastoma and PNET cell lines and tumors.

The stem cell factor/kit tyrosine kinase receptor pathway is related to tumor growth and progression in several cancers including Ewing sarcoma, a peripheral PNET (pPNET). Identifying additional groups of tumors that may use the pathway is important as they might be responsive to imatinib mesylate treatment. MB and central PNET (cPNET) are embryonal tumors of the CNS that share similar undifferentiated morphology with Ewing sarcomas and display aggressive clinical behavior. cPNET outcome is significantly lower than MB outcome, even for localized tumors treated with high-risk MB therapy. The elucidation of signaling pathways involved in MB and cPNET pathogenesis, and the discovery of new therapeutic targets is necessary to improve the treatment of these neoplasms. We analyzed KIT expression in 2 MB, one pPNET, one cPNET and 2 rhabdomyosarcoma (RMS) cell lines. Also, in 13 tumor samples (12 MB and one cPNET), we found KIT overexpression in the most aggressive cell lines (metastatic MB and pPNET). Hypermethylation of KIT was clear in the RMS non-expressing cell lines. Among MB tumors, we could see variable levels of KIT expression; a subset of them (25%) might be related in its growth pattern to KIT up-regulation. No methylated KIT was detected in the tumors expressing the lowest levels of KIT. Our results point to methylation as an epigenetic regulatory mechanism for KIT inhibition only in the KIT non-expressing RMS cell lines, and neither in the rest of the cell lines nor in the tumor samples.