ABSTRACT
The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 Å resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.
Subject(s)
Colorectal Neoplasms , GTP Phosphohydrolases , MAP Kinase Signaling System , Membrane Proteins , Mutation , Proto-Oncogene Proteins p21(ras) , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Crystallography, X-Ray , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , HEK293 Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Domains , Proto-Oncogene Mas , Proto-Oncogene Proteins p21(ras)/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Exome Sequencing , raf Kinases/genetics , raf Kinases/metabolismABSTRACT
OBJECTIVES: Current protocols for processing multiple prostate biopsy cores per case are uneconomical and cumbersome. Tissue fragmentation and loss compromise cancer diagnosis. We sought to study an alternate method to improve processing and diagnosis of prostate cancer. METHODS: Two sets of sextant biopsy specimens from near-identical locations were obtained ex vivo from 48 prostate specimens. One set was processed in the standard fashion while the other was processed using the BxChip, a proprietary biomimetic matrix that accommodates six cores on a single chip. Parameters including grossing, embedding, sectioning and reading time, length of tissue, and degree of fragmentation were compared. RESULTS: A significant reduction (more than threefold) in preanalytical and analytical time was observed using the multiplex method. Nonlinear fragmentation was absent, in contrast to standard processing. CONCLUSIONS: The BxChip reduced tissue fragmentation and increased efficiency of prostate biopsy diagnosis. It also resulted in overall cost savings and significantly increased tissue length.
