Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer. Inflammation is a typical feature in cSCC progression. Analysis of the expression of inflammasome components in cSCC cell lines and normal human epidermal keratinocytes revealed upregulation of the expression of AIM2 mRNA and protein in cSCC cells. Elevated levels of AIM2 mRNA were noted in cSCCs in vivo compared with normal skin. Strong and moderate tumor cell specific expression of AIM2 was detected with immunohistochemistry (IHC) in sporadic human cSCCs in vivo, whereas expression of AIM2 was moderate in cSCC in situ (cSCCIS) and low or absent in actinic keratosis (AK) and normal skin. IHC of cSCCs, cSCCIS and AKs from organ transplant recipients also revealed strong and moderate tumor cell specific expression of AIM2 in cSCCs. Knockdown of AIM2 resulted in reduction in viability of cSCC cells and onset of apoptosis. RNA-seq and pathway analysis after knockdown of AIM2 in cSCC cells revealed downregulation of the biofunction category Cell cycle and upregulation of the biofunction category Cell Death and Survival. Knockdown of AIM2 also resulted in reduction in invasion of cSCC cells and downregulation in production of invasion proteinases MMP1 and MMP13. Knockdown of AIM2 resulted in suppression of growth and vascularization of cSCC xenografts in vivo. These results provide evidence for the role of AIM2 in the progression of cSCC and identify AIM2 inflammasome function as a potential therapeutic target in these invasive and metastatic tumors.

Keratinocyte-derived skin cancer, cutaneous squamous cell carcinoma (cSCC), is the most common metastatic skin cancer. We have examined the role of Eph/ephrin signaling in the progression of cSCC. Analysis of the expression of EPH and EFN families in cSCC cells and normal epidermal keratinocytes revealed overexpression of EPHB2 mRNA in cSCC cells and cSCC tumors in vivo. Tumor cell-specific overexpression of EphB2 was detected in human cSCCs and in chemically induced mouse cSCCs with immunohistochemistry, whereas the expression of EphB2 was low in premalignant lesions and normal skin. Knockdown of EphB2 expression in cSCC cells suppressed growth and vascularization of cSCC xenografts in vivo and inhibited proliferation, migration, and invasion of cSCC cells in culture. EphB2 knockdown downregulated expression of genes associated with biofunctions cell viability, migration of tumor cells, and invasion of tumor cells. Among the genes most downregulated by EphB2 knockdown were MMP1 and MMP13. Moreover, activation of EphB2 signaling by ephrin-B2-Fc enhanced production of invasion proteinases matrix metalloproteinase-13 (MMP13) and MMP1, and invasion of cSCC cells. These findings provide mechanistic evidence for the role of EphB2 in the early progression of cSCC to the invasive stage and identify EphB2 as a putative therapeutic target in this invasive skin cancer.