Tenascin-C (TNC) is highly expressed in melanoma; however, little is known about its functions. Recent studies indicate that TNC plays a role within the stem cell niche. We hypothesized that TNC creates a specific environment for melanoma cells to exhibit a stem cell-like phenotype, driving tumor growth and evading conventional therapies. TNC expression was strongly up-regulated in melanoma cells grown as 3D spheres (enriched for stem-like cells) when compared to adherent cells. Down-modulation of TNC by shRNA-lentiviruses significantly decreased the growth of melanoma spheres. The incidence of pulmonary metastases after intravenous injection of TNC knockdown cells was significantly lower in NOD/SCID IL2Rγnull mice compared to control cells. Melanoma spheres contain and increased number of side population (SP) cells, which exhibited stem cell characteristics and the potential for drug resistance due to their high efflux capacity. Knockdown of TNC dramatically decreased the SP fraction in melanoma spheres and lowered their resistance to doxorubicin treatment, likely due to the down-regulation of multiple ABC transporters, including ABCB5. These data suggest that TNC plays a critical role in melanoma progression by mediating protective signals in the therapy-resistant population of melanoma.

Although cyclin dependent kinase (CDK)-2 is known to be dispensable for the growth of most tumors, it is thought to be important for the proliferation of melanoma cells, where its expression is controlled by the melanocyte-lineage specific transcription factor MITF. Treatment of a panel of melanoma cells with the CDK inhibitor dinaciclib led to a concentration-dependent inhibition of growth under both 2D adherent and 3D organotypic cell culture conditions. Dinaciclib targeted melanoma cell lines regardless of cdk2 or MITF levels. Inhibition of growth was associated with a rapid induction of G2/M cell arrest and apoptosis. Treatment of human melanoma mouse xenografts with dinaciclib led to tumor regression associated with reduced retinoblastoma protein phosphorylation and Bcl-2 expression. Further mechanistic studies revealed that dinaciclib induces p53 expression whilst simultaneously downregulating the expression of the anti-apoptotic factors Mcl-1 and XIAP. To clarify the role of p53 activation in the dinaciclib-induced cell death, we generated melanoma cell lines in which p53 expression was knocked down using a shRNA lentiviral vector. Knockdown of p53 completely abolished the induction of apoptosis seen following dinaciclib treatment as shown by a lack of annexin-V staining and caspase-3 cleavage. Altogether, these data show that dinaciclib induces apoptosis in a large panel of melanoma cell lines through a mechanism requiring p53 expression.

A major barrier to curative therapy for most malignancies is intrinsic drug resistance within discrete tumor cell subpopulations, which are presently ill-defined. Previous studies show that carcinoma cell lines abundantly expressing mesenchymal markers have increased resistance to both conventional chemotherapy and epidermal growth factor receptor (EGFR)-targeted agents. However it is unclear whether small mesenchymal-like subpopulations contribute to therapy resistance in individual carcinomas with predominantly epithelial features. Here we identified a mesenchymallike subset expressing low E-cadherin (Ecad lo) and high vimentin (Vim hi) within human squamous cell carcinomas of the esophagus (ESCC) and head and neck (HNSCC). This minority subset was readily isolated from two HNSCC lines (SCC9 and OCTT2) and was identifiable in vivo, not only in mouse xenografts of these lines but also in the clinical specimen from which a HNSCC cell line (OCTT2) was derived. The minority Ecad lo/Vim hi subset contained more low-turnover cells and was less proliferative overall, correlating with increased resistance to the conventional chemotherapeutic agent paclitaxel. The Ecad lo/Vim hi subpopulation also demonstrated diminished activity in the MAP kinase/PI3-kinase pathways, likely arising from decreased EGFR expression and correlating with increased in vitro resistance to the EGFR-targeted agent, cetuximab. This mesenchymal-like subpopulation displayed comparable drug resistancein vivo, becoming enriched by cetuximab treatment in a mouse xenograft derived directly from a clinical HNSCC specimen. Furthermore, the Ecad lo/Vim hi and Ecad hi/Vim lo subsets were dynamic and reversible in their phenotypes, showing the capacity to repopulate each other from single cell clones. Taken together, these results show that a low-turnover, mesenchymal-like subpopulation in squamous cell carcinomas has diminished EGFR pathway function and elevated intrinsic resistance to both conventional and targeted chemotherapies. They further suggest that this mesenchymal-like subset retains the plasticity to repopulate other malignant subpopulations post therapy. Citation Information: Cancer Res 2009;69(23 Suppl):C77.