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Aberrant regulation of growth signaling is a hallmark of cancer development that often occurs through the constitutive activation of growth factor receptors or their downstream effectors. Using validation-based insertional mutagenesis (VBIM), we identified family with sequence similarity 83, member B (FAM83B), based on its ability to substitute for RAS in the transformation of immortalized human mammary epithelial cells (HMECs). We found that FAM83B coprecipitated with a downstream effector of RAS, CRAF. Binding of FAM83B with CRAF disrupted CRAF/14-3-3 interactions and increased CRAF membrane localization, resulting in elevated MAPK and mammalian target of rapamycin (mTOR) signaling. Ablation of FAM83B inhibited the proliferation and malignant phenotype of tumor-derived cells or RAS-transformed HMECs, implicating FAM83B as a key intermediary in EGFR/RAS/MAPK signaling. Analysis of human tumor specimens revealed that FAM83B expression was significantly elevated in cancer and was associated with specific cancer subtypes, increased tumor grade, and decreased overall survival. Cumulatively, these results suggest that FAM83B is an oncogene and potentially represents a new target for therapeutic intervention. IntroductionThe discovery of targets suitable for the development of specific and effective anticancer therapies remains one of the principal challenges facing cancer research. The identification of genes involved in tumorigenesis is essential for devising new targeted therapeutics and can be greatly facilitated by phenotypic-based forward genetic screens for mutations contributing to malignant transformation in human cell models. We recently created a validation-based insertional mutagenesis (VBIM) strategy that expands the application of reversible promoter insertion to nearly any type of mammalian cell (1). The VBIM strategy uses the unique transcriptomes of different human epithelial cell types and provides opportunities for the identification of tissue-specific oncogenes and tumor suppressors. The VBIM lentiviruses alter the unique transcriptome of the model system by introducing promoters into the genome, resulting in dominant genetic alterations that increase the expression of sequences neighboring the insertion sites. By using Cre recombinase-mediated excision of the VBIM promoter, one can revert the VBIM-specific mutants and distinguish them from spontaneous mutants, allowing spontaneous mutants to be eliminated from further study.We have used the VBIM strategy to identify family with sequence similarity 83, member B (FAM83B), as a putative oncogene capable of promoting the transformation of immortalized human mammary epithelial cells (HMECs). We demonstrated that elevated FAM83B expression stimulated aberrant activation of MAPK signaling by altering binding of regulatory 14-3-3 proteins to CRAF and increasing CRAF membrane localization. In addition to driving cellular transformation, FAM83B mRNA was significantly elevated in many human tumor tissues. Ablation of FAM83B from breast cancer cells with ele...

Metabolic reprogramming is a hallmark of cancer. The changes in metabolism are adaptive to permit proliferation, survival, and eventually metastasis in a harsh environment. Stable isotope-resolved metabolomics (SIRM) is an approach that uses advanced approaches of NMR and mass spectrometry to analyze the fate of individual atoms from stable isotope-enriched precursors to products to deduce metabolic pathways and networks. The approach can be applied to a wide range of biological systems, including human subjects. This review focuses on the applications of SIRM to cancer metabolism and its use in understanding drug actions.Metabolism is the collection of predominantly enzyme-catalyzed biochemical transformations that meet the growth and survival demands of an organism. For nearly a century, scientists have documented profound metabolic changes that occur in tumors (1, 2). One of the earliest insights into cancer metabolism came when Otto Warburg noted increased uptake and fermentation of glucose (Glc) 3 to lactate in tumors relative to surrounding tissue, even in the presence of ample oxygen (2). Clinical cancer diagnosis exploits this "Warburg effect" by measuring uptake of the Glc analogue 18 F-deoxyglucose using positron emission tomography (PET), as the metabolic demands of differentiated, non-proliferating tissues generally require far less Glc than tumors (3).Oncoproteins and tumor suppressors are well-established regulators of metabolism, and mutations or dysregulated expression can lead to the altered metabolic phenotypes observed in many cancers (4, 5). Inactivating mutations in enzymes such as fumarate hydratase (FH) or succinate dehydrogenase (SDH) induce pathophysiological accumulation of substrates that inhibit other critical enzyme functions, whereas less common gain-of-function mutations such as in isocitrate dehydrogenases (IDH) produce metabolites that directly deregulate cellular processes (6). Additionally, cancer cells frequently express fetal isoforms of metabolic enzymes that are subject to different regulatory mechanisms to provide growth advantages (7). Gaining a systematic understanding of the metabolic changes that occur during carcinogenesis can thus be exploited for therapeutic and diagnostic purposes.Stable isotope-resolved metabolomics (SIRM) is a powerful approach developed by others and by us where isotopically enriched precursors such as [ 13 C 6 ]Glc are administered to a biological system, and the ensuing metabolic transformations are determined using appropriate analytic techniques to enable robust reconstruction of metabolic pathways (8 -11). Stable isotopes are non-radioactive and in SIRM practice are identical chemically and functionally to the most abundant isotope of that element. Incorporating stable isotopes such as 2 H, 13 C, or 15 N into biological precursors has long been used to trace their metabolism in living systems (12). SIRM is thus distinct from non-tracer-based metabolomics profiling for statistical models. Here, we review SIRM applications and demonstrate h...