The therapeutic responses of many solid tumours to chemo- and radio-therapies are far from fully effective but therapies targeting malignancy-related cellular changes show promise for further control. In head and neck squamous cell carcinoma, the epidermal growth factor receptor (EGFR) is commonly overexpressed and investigation of agents that block this receptor indicate a limited response when used alone but an ability to enhance the actions of other drugs. The hierarchical stem cell patterns present in tumours generate cellular heterogeneity and this is further complicated by cancer stem cells (CSC) shifting between epithelial (Epi-CSC) and mesenchymal (EMT-CSC) states. To clarify how such heterogeneity influences responses to EGFR blocking, we examined the effects of Cetuximab and Erlotinib on the cell sub-populations in HNSCC cell lines. These agents reduced cell proliferation for all subpopulations but induced little cell death. They did however induce large shifts of cells between the EMT-CSC, Epi-CSC and differentiating cell compartments. Loss of EMT-CSCs reduced cell motility and is expected to reduce invasion and metastasis. EGFR blocking also induced shifts of Epi-CSCs into the differentiating cell compartment which typically has greater sensitivity to chemo/radiation, an effect expected to enhance the overall response of tumour cell populations to adjunctive therapies.
The supramolecular presentation of extracellular matrix components on surfaces provides a platform for the investigation and control of cell behavior. Hyaluronan (HA) is one of the main components of the extracellular environment and has been shown to play an important role in different cancers and their progression. However, current methods of HA immobilization often require its chemical modification. Herein, a peptide‐based self‐assembled monolayer (SAM) is used as an anchor to immobilize unmodified HA on a bare gold surface, as demonstrated by the quartz crystal microbalance with dissipation monitoring. Peptide‐HA surfaces show increased roughness and greater hydrophobicity when compared to poly‐D‐lysine/HA surfaces, as measured by atomic force microscopy and water contact angle, respectively. Additionally, the peptide SAM can be micro‐contact printed and used to restrict the presentation of HA to specific regions, thereby creating HA patterned surfaces to examine cell behavior. When used for cell culture, these surfaces result in altered adhesion and migration of LUC4 head and neck squamous cell carcinoma cells. These biomimetic surfaces can provide insights into the role of HA in cancer and other diseases and be used as a platform for the development of cell sorting devices.
Small-molecule inhibitors of the Hedgehog (HH) pathway receptor Smoothened (SMO) have been effective in treating some patients with basal cell carcinoma (BCC), where the HH pathway is often activated, but many patients respond poorly. In this study, we report the results of investigations on PTCH1 signaling in the HH pathway that suggest why most patients with BCC respond poorly to SMO inhibitors. In immortalized human keratinocytes, PTCH1 silencing led to the generation of a compact, holoclone-like morphology with increased expression of SMO and the downstream HH pathway transcription factor GLI1. Notably, although siRNA silencing of SMO in PTCH1-silenced cells was sufficient to suppress GLI1 activity, this effect was not phenocopied by pharmacologic inhibition of SMO, suggesting the presence of a second undefined pathway through which SMO can induce GLI1. Consistent with this possibility, we observed increased nuclear localization of SMO in PTCH1-silenced cells as mediated by a putative SMO nuclear/nucleolar localization signal [N(o)LS]. Mutational inactivation of the N(o)LS ablated this increase and suppressed GLI1 induction. Immunohistologic analysis of human and mouse BCC confirmed evidence of nuclear SMO, although the pattern was heterogeneous between tumors. In PTCH1-silenced cells, >80% of the genes found to be differentially expressed were unaffected by SMO inhibitors, including the putative BCC driver gene CXCL11. Our results demonstrate how PTCH1 loss results in aberrant regulation of SMO-independent mechanisms important for BCC biology and highlights a novel nuclear mechanism of SMO-GLI1 signaling that is unresponsive to SMO inhibitors. This study describes novel noncanonical Hedgehog signaling, where SMO enters the nucleus to activate GLI1, a mode that is unaffected by SMO inhibitors, thus prompting re-evaluation of current BCC treatment as well as new potential therapies targeting nuclear SMO. .
Background Cancer cell plasticity, as seen in epithelial-to-mesenchymal transition, can lead to metastasis and therapeutic failure. Another cell type, the amoeboid, has now been isolated in oral cancer. Previously seen in, but not isolated from melanomas and sarcomas, it has been associated with poorer prognosis. The aim of the study was to investigate the role of the amoeboid cell in oral cancer, with the hypothesis that it is a plastic, but more invasive and chemoresistant, cell type. MethodsIn this in-vitro study of oral cancer cell lines (n=6), fresh tumour specimens, and mice, we used high-throughput invasion assays, migration and drug response assays, protein and gene expression profi ling, mechanistic and pathway analysis, fl uorescence-activated cell sorting, gene knockdowns, and immunostaining. At least three cell lines were used for each laboratory technique, with a minimum of triplicate repeats and appropriate statistical analysis.Findings In all cell lines, amoeboid cells were smaller than both epithelial and mesenchymal cells (median cell area 295 μm² [IQR 218-399] vs 884 [573-1281] vs 598 [413-815]) but were signifi cantly more migratory with a mean velocity of 1•1 μm/min (SD 0•2) versus 0•16 (0•08) for mesenchymal cells (p<0•0001). Amoeboid cells were four to 20 times more invasive than other cell types (p<0•0001). Greater chemoresistance was demonstrated against common agents including paclitaxel and etoposide. Gene analysis produced signatures associated with angiogenesis, anti-apoptosis, and invasion. Stemness genes were upregulated. Plasticity between phenotypes was clearly seen.Interpretation We describe a new amoeboid cell phenotype, which is derived from epithelial cancer cells, that might confer upon carcinomas a greater ability to invade, disseminate, and resist therapy. A switch to an amoeboid phenotype could be a useful escape strategy for cancers, providing them with alternative modes for migration and invasion. However, this cell type essentially lacks keratin, which could complicate histopathological assessment of tumour spread. Pathway elucidation might yield new potential amoeboid targets. Targeting amoeboid cells, which may now become possible with their isolation and analysis, could be essential to improve patient outcomes.
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