Purpose Aggressive cutaneous squamous cell carcinoma (cSCC) is often a disfiguring and lethal disease. Very little is currently known about the mutations that drive aggressive cSCC. Experimental Design Whole exome sequencing was performed on 39 cases of aggressive cSCC to identify driver genes and novel therapeutic targets. Significantly mutated genes were identified with MutSig or complementary methods developed to specifically identify candidate tumor suppressors based upon their inactivating mutation bias. Results Despite the very high mutational background caused by UV exposure, 23 candidate drivers were identified including the well-known cancer-associated genes TP53, CDKN2A, NOTCH1, AJUBA, HRAS, CASP8, FAT1, and KMT2C (MLL3). Three novel candidate tumor suppressors with putative links to cancer or differentiation, NOTCH2, PARD3 and RASA1, were also identified as possible drivers in cSCC. KMT2C mutations were associated with poor outcome and increased bone invasion. Conclusions The mutational spectrum of cSCC is similar to that of head and neck squamous cell carcinoma and dominated by tumor suppressor genes. These results improve the foundation for understanding this disease and should aid in identifying and treating aggressive cSCC.

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH2-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.

Purpose The objective of this study was to determine the incidence of the MYB-MFIB fusion in salivary adenoid cystic carcinoma (ACC), to establish the clinicopathological significance of the fusion and to analyze the expression of MYB in ACCs in the context of the MYB-NFIB fusion. Experimental Design We performed an extensive analysis involving 123 cancers of the salivary gland, including primary and metastatic ACCs, and non-ACC salivary carcinomas. MYB-NFIB fusions were identified by reverse transcription-PCR (RT-PCR) and sequencing of the RT-PCR products, and confirmed by fluorescence in situ hybridization. MYB RNA expression was determined by quantitative RT-PCR and protein expression was analyzed by immunohistochemistry. Results The MYB-NFIB fusion was detected in 28% primary and 35% metastatic ACCs, but not in any of the non-ACC salivary carcinomas analyzed. Different exons in both MYB and NFIB genes were involved in the fusions, resulting in expression of multiple chimeric variants. Notably, MYB was overexpressed in the vast majority of the ACCs, although MYB expression was significantly higher in tumors carrying the MYB-NFIB fusion. The presence of the MYB-NFIB fusion was significantly associated (p = 0.03) with patients older than 50 years of age. No correlation with other clinicopathological markers, factors and survival was found. Conclusions We conclude that the MYB-NFIB fusion characterizes a subset of ACCs and contributes to MYB overexpression. Additional mechanisms may be involved in MYB overexpression in ACCs lacking the MYB-NFIB fusion. These findings suggest that MYB may be a specific novel target for tumor intervention in patients with ACC.

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Tumor necrosis factor (TNF) is a cytokine produced by macrophages and T lymphocytes that acts through two distinct receptors, TNFR1 (60 kD, CD120a) and TNFR2 (80 kD, CD120b), to affect cellular proliferation, differentiation, survival, and cell death. In addition to its proinflammatory actions in mucosal tissue, TNF is important for liver regeneration. Keratin 8 (K8) and keratin 18 (K18) form intermediate filaments characteristic of liver and other single cell layered, internal epithelia and their derivative cancers. K8-deficient (K8−) mice, which escape embryonic lethality, develop inflammatory colorectal hyperplasia, mild liver abnormalities, and tolerate hepatectomy poorly. We show that normal and malignant epithelial cells deficient in K8 and K18 are ∼100 times more sensitive to TNF-induced death. K8 and K18 both bind the cytoplasmic domain of TNFR2 and moderate TNF-induced, Jun NH2-terminal kinase (JNK) intracellular signaling and NFκB activation. Furthermore, K8− and K18− mice are much more sensitive to TNF dependent, apoptotic liver damage induced by the injection of concanavalin A. This moderation of the effects of TNF may be the fundamental function of K8 and K18 common to liver regeneration, inflammatory bowel disease, hepatotoxin sensitivity, and the diagnostic, persistent expression of these keratins in many carcinomas.