Human papillomavirus negative (HPV-) head and neck squamous cell carcinomas (HNSCC) are deadly and common cancers. Recent genomic studies implicate multiple genetic pathways including cell-signalling, cell-cycle and/or immune evasion in their development. Here, we analyze public datasets and uncover a previously unappreciated role of epigenome deregulation in the genesis of 13% HPV-HNSCCs. Specifically, we identify novel recurrent p.K36M mutations occurring in multiple histone H3 genes. We further validate their presence in multiple independent HNSCC datasets and show that along with previously described NSD1 mutations, they correspond to a specific DNA methylation cluster. H3K36M and NSD1 defects converge on altering H3K36 methylation, subsequently blocking cellular differentiation and promoting oncogenesis. Our data further indicate surprisingly limited redundancy for NSD family members in HPV-HNSCCs and suggest a potential role of impaired H3K36 methylation in their development. Further investigation of drugs targeting chromatin regulators is warranted in HPV-HNSCCs driven by aberrant H3K36 methylation.

Tumor-initiating cells (TICs) are defined by their ability to form tumors after xenotransplantation in immunodeficient mice and appear to be relatively rare in most human cancers. Recent data in melanoma indicate that the frequency of TICs increases dramatically via more permissive xenotransplantation conditions, raising the possibility that the true frequency of TICs has been greatly underestimated in most human tumors. We compared the growth of human pancreatic, non-small cell lung, and head and neck carcinomas in NOD/SCID and NSG mice. Although TIC frequency was detected up to 10-fold higher in NSG mice, it remained low (<1 in 2500 cells) in all cases. Moreover, aldehyde dehydrogenase-positive (ALDH(+)) and CD44(+)CD24(+) cells, phenotypically distinct cells enriched in TICs, were equally tumorigenic in NOD/SCID and NSG mice. Our findings demonstrate that TICs are rare in these cancers and that the identification of TICs and their frequency in other human malignancies should be validated via primary tumors and highly permissive xenotransplantation conditions.

Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ADAM10. Exosomal ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase RhoA. Moreover, ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ADAM10-rich exosomes that promote cell motility and activate RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression.