Tumour progression involves the alteration of many regulatory pathways. Current theories of tumour progression have focused on clonal populations of cells that undergo successive genetic alterations and sequential selection of dominant clones. However, tumour progression through lineal evolution is inefficient. Multiclonality has been observed in many cancers. The presence of more than one clone in the tumour provides an opportunity for interaction, which may facilitate the tumour progression through interclonal cooperativity.
To evaluate whether interclonal cooperativity plays a major role in the progression of HNSCC, we stably overexpressed Snail2 (epithelial-mesenchymal transition [EMT] gene), and Hras (oncogenic G12V mutant) with distinct fluorescent protein tags, in the HNSCC cell line HN13. Subcutaneous injection of HN13 cells with overexpression of Hras formed primary tumours in immunodeficient athymic nude mice. However, no Hras-expressing cells were detected in the draining lymph nodes. In contrast, Snail2- expressing HN13 cells did not form primary tumours in nude mice but Snail2 cells were detected in the lymph nodes. Additionally, injection of a mixed population of Hras and Snail2- expressing HN13 cells formed tumours containing both cell types and Snail2 cells migrated to lymph nodes.
To better understand the mechanisms of Hras-mediated tumorigenesis, we expressed Hras double mutants that selectively activate downstream signaling pathways. HN13 cells overexpressing Hras (G12V, E37G) double mutant cells formed primary tumours in nude mice, whereas HN13 cells expressing Hras (G12V, T35S) or Hras (G12V, Y40C) double mutants did not form tumours. The in vitro proliferation assay demonstrated that proliferation of Hras double mutant (G12V, E37G) and single mutant (G12V) cells was enhanced compared to Hras double mutants (G12V, T35S and G12V, Y40C) cells. Ongoing studies on combinations of different Hras double mutant expressing cells together with Snail2/Hras results will elucidate the role of clonal interactions and tumour progression in HNSCC.