CRISPR-Cas9-based target validation for p53-reactivating model compounds.

Inactivation of the p53 tumor suppressor by Mdm2 is one of the most frequent events in cancer, so compounds targeting the p53-Mdm2 interaction are promising for cancer therapy. Mechanisms conferring resistance to p53-reactivating compounds are largely unknown. Here we show using CRISPR-Cas9-based target validation in lung and colorectal cancer that the activity of nutlin, which blocks the p53-binding pocket of Mdm2, strictly depends on functional p53. In contrast, sensitivity to the drug RITA, which binds the Mdm2-interacting N terminus of p53, correlates with induction of DNA damage. Cells with primary or acquired RITA resistance display cross-resistance to DNA crosslinking compounds such as cisplatin and show increased DNA cross-link repair. Inhibition of FancD2 by RNA interference or pharmacological mTOR inhibitors restores RITA sensitivity. The therapeutic response to p53-reactivating compounds is therefore limited by compound-specific resistance mechanisms that can be resolved by CRISPR-Cas9-based target validation and should be considered when allocating patients to p53-reactivating treatments.

High expression of class III ß-tubulin in upper gastrointestinal cancer types.

Class III ß-tubulin (TUBB3) is a component of microtubules of neuronal cells that is upregulated in various cancer entities. To better understand the role of TUBB3 in upper gastrointestinal tract cancer types, the present study assessed TUBB3 expression in tissue microarrays including 189 gastric and 428 esophageal cancer. TUBB3 expression was detected in 62.4% of gastric cancer, 73.8% of esophageal adenocarcinoma and 88.7% of esophageal squamous cell cancer, while control samples of normal esophageal and gastric epithelium were TUBB3-negative. TUBB3 positivity was not associated with the International Union Against Cancer classification, World Health Organization grading, lymph node involvement or distant metastasis in any entity. Of note, TUBB3 expression was associated with tumor localization and prognosis in gastric cancer, with the tumor stage in esophageal adenocarcinoma, and with the resection margin in esophageal squamous cell cancer. In conclusion, the substantial rate of positivity for TUBB3 already in early stages of gastric cancer in combination with the lack of a further increase in frequency with tumor stage, may suggest, that TUBB3 upregulation is rather relevant for cancer development than for cancer progression. TUBB3 might be a suitable prognostic biomarker in gastric cancer types.