Deep Proteomics of Breast Cancer Cells Reveals that Metformin Rewires Signaling Networks Away from a Pro-growth State.

Metformin is the most frequently prescribed drug for type 2 diabetes. In addition to its hypoglycemic effects, metformin also lowers cancer incidence. This anti-cancer activity is incompletely understood. Here, we profiled the metformin-dependent changes in the proteome and phosphoproteome of breast cancer cells using high-resolution mass spectrometry. In total, we quantified changes of 7,875 proteins and 15,813 phosphosites after metformin changes. To interpret these datasets, we developed a generally applicable strategy that overlays metformin-dependent changes in the proteome and phosphoproteome onto a literature-derived network. This approach suggested that metformin treatment makes cancer cells more sensitive to apoptotic stimuli and less sensitive to pro-growth stimuli. These hypotheses were tested in vivo; as a proof-of-principle, we demonstrated that metformin inhibits the p70S6K-rpS6 axis in a PP2A-phosphatase dependent manner. In conclusion, analysis of deep proteomics reveals both detailed and global mechanisms that contribute to the anti-cancer activity of metformin.

SIGNOR: a database of causal relationships between biological entities.

Assembly of large biochemical networks can be achieved by confronting new cell-specific experimental data with an interaction subspace constrained by prior literature evidence. The SIGnaling Network Open Resource, SIGNOR (available on line at http://signor.uniroma2.it), was developed to support such a strategy by providing a scaffold of prior experimental evidence of causal relationships between biological entities. The core of SIGNOR is a collection of approximately 12,000 manually-annotated causal relationships between over 2800 human proteins participating in signal transduction. Other entities annotated in SIGNOR are complexes, chemicals, phenotypes and stimuli. The information captured in SIGNOR can be represented as a signed directed graph illustrating the activation/inactivation relationships between signalling entities. Each entry is associated to the post-translational modifications that cause the activation/inactivation of the target proteins. More than 4900 modified residues causing a change in protein concentration or activity have been curated and linked to the modifying enzymes (about 351 human kinases and 94 phosphatases). Additional modifications such as ubiquitinations, sumoylations, acetylations and their effect on the modified target proteins are also annotated. This wealth of structured information can support experimental approaches based on multi-parametric analysis of cell systems after physiological or pathological perturbations and to assemble large logic models.

Metformin Induces Apoptosis and Downregulates Pyruvate Kinase M2 in Breast Cancer Cells Only When Grown in Nutrient-Poor Conditions.

INTRODUCTION: Metformin is proposed as adjuvant therapy in cancer treatment because of its ability to limit cancer incidence by negatively modulating the PI3K/AKT/mTOR pathway. In vitro, in addition to inhibiting cancer cell proliferation, metformin can also induce apoptosis. The molecular mechanism underlying this second effect is still poorly characterized and published data are often contrasting. We investigated how nutrient availability can modulate metformin-induced apoptosis in three breast cancer cell lines. MATERIAL AND METHODS: MCF7, SKBR3 and MDA-MB-231 cells were plated in MEM medium supplemented with increasing glucose concentrations or in DMEM medium and treated with 10 mM metformin. Cell viability was monitored by Trypan Blue assay and treatment effects on Akt/mTOR pathway and on apoptosis were analysed by Western Blot. Moreover, we determined the level of expression of pyruvate kinase M2 (PKM2), a well-known glycolytic enzyme expressed in cancer cells. RESULTS: Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium. Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation. Finally, we showed that, in nutrient-poor conditions, metformin was able to modulate the intracellular glycolytic equilibrium by downregulating PKM2 expression and that this mechanism was mediated by AMPK activation. CONCLUSION: We demonstrated that metformin induces breast cancer cell apoptosis and PKM2 downregulation only in nutrient-poor conditions. Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect. These data demonstrate that the reduction of nutrient supply in tumors can increase metformin efficacy and that modulation of PKM2 expression/activity could be a promising strategy to boost metformin anti-cancer effect.

p53 mediates TNF-induced epithelial cell apoptosis in IBD.

Chronic ulcerative colitis (CUC) is characterized by increased intestinal epithelial cell (IEC) apoptosis associated with elevated tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), and p53. We previously showed that p53 is increased in crypt IECs in human colitis and is needed for IEC apoptosis in chronic dextran sulfate sodium-colitis. Herein, we examined the roles of TNF and iNOS in regulating p53-induced IEC apoptosis in CUC. The IEC TUNEL staining, caspases 3, 8, and 9, and p53 protein levels, induced by anti-CD3 monoclonal antibody (mAb) activation of T cells, were markedly reduced in TNF receptor 1 and 2 gene knockout mice. Induction of IEC apoptosis correlated with increased p53, which was attenuated in iNOS(-/-) mice. IEC p53 levels and apoptosis were reduced in IL-10(-/-) colitic mice treated with neutralizing TNF mAb and the iNOS inhibitor, aminoguanidine, further suggesting that TNF and iNOS are upstream of p53 during colitis-induced IEC apoptosis. IEC apoptosis and p53 levels were assessed in control versus untreated or anti-TNF-treated CUC patients with equivalent levels of inflammation. Data indicated that IEC apoptosis and p53 levels were clearly higher in untreated CUC but markedly reduced in patients treated with anti-TNF mAb. Therefore, TNF-induced iNOS activates a p53-dependent pathway of IEC apoptosis in CUC. The inhibition of IEC apoptosis may be an important mechanism for mucosal healing in anti-TNF-treated CUC patients.

Combined analysis of galectin-3 and BRAFV600E improves the accuracy of fine-needle aspiration biopsy with cytological findings suspicious for papillary thyroid carcinoma.

Ten to fifteen percent of fine-needle aspiration biopsy (FNAB) of thyroid nodules are indeterminate. Galectin-3 (Gal-3) and the oncogene BRAFV600E are markers of malignancy useful to improve FNAB accuracy. The objective of this study was to determine whether the combined analysis of Gal-3 and BRAFV600E expression in thyroid aspirates could improve the diagnosis in FNAB with suspicious cytological findings. Two hundred and sixty-one surgical thyroid tissues and one hundred and forty-four thyroid aspirates were analyzed for the presence of the two markers. In surgical specimens, Gal-3 expression was present in 27.4% benign nodules, 91.9% papillary (PTC) and 75% follicular (FTC) thyroid carcinomas. BRAFV600E was not detected in 127 benign nodules, as well as in 32 FTCs, while was found in 42.9% PTC. No correlation was found between BRAF mutation and Gal-3 expression. Forty-seven consecutive FNAB suspicious for PTC were analyzed for the presence of the two markers. Of these nodules, 23 were benign at histology, 6 were positive for Gal-3, none displayed BRAFV600E, and 17 were negative for both the markers. Twenty suspicious nodules were diagnosed as PTC and four FTCs at histology. Of these 24 carcinomas, 9 resulted positive for BRAFV600E, 17 for Gal-3, and 22 for one or both the markers. The sensitivity, specificity, and accuracy for the presence of Gal-3 and/or BRAFV600E were significantly higher than those obtained for the two markers alone. Notably, the negative predictive value increased from 70.8 to 89.5%. In conclusion, the combined detection of Gal-3 and BRAFV600E improves the diagnosis in FNAB with cytological findings suspicious for PTC and finds clinical application in selected cases.

Detection of RET/PTC, TRK and BRAF mutations in preoperative diagnosis of thyroid nodules with indeterminate cytological findings.

BACKGROUND: Fine-needle aspiration biopsy (FNAB) is the primary means to distinguish benign from malignant nodules and select patients for surgery. However, adjunctive diagnostic tests are needed because in 20-40% of cases the FNAB result is uncertain. OBJECTIVE: We investigated whether a search for the oncogenes RET/PTC, TRK and BRAF(V600E) in thyroid aspirates could refine an uncertain diagnosis. PATIENTS AND METHODS: A total of 132 thyroid aspirates, including colloid nodules, inadequate samplings, indeterminate and suspicious for malignancy were analysed by reverse transcription polymerase chain reaction (RT-PCR) and mutant allele-specific amplification techniques for the presence of oncogenes. RESULTS: No oncogenes were detected in 48 colloid nodules, 46 inadequate and 19 indeterminate FNABs, then confirmed to be benign at histology. No oncogenes were detected in one follicular thyroid cancer (FTC) with indeterminate cytology. Five out of six papillary thyroid cancers (83%) with FNAB suspicious for malignancy were correctly diagnosed by the presence of oncogenes. Among these, four (67%) contained the BRAF mutation and one (17%) contained RET/PTC-3. On final analysis, no false-positive results were reported in 131 samples and five out of seven carcinomas (71%) were correctly diagnosed. The finding of oncogenes in FNAB specimens suspicious for malignancy guided the extent of surgical resection, changing the surgery from diagnostic to therapeutic in five cases. CONCLUSIONS: Detection of RET/PTC, TRK and BRAF(V600E) in FNAB specimens is proposed as a diagnostic adjunctive tool in the evaluation of thyroid nodules with suspicious cytological findings.

Detection of BRAF mutation in thyroid papillary carcinomas by mutant allele-specific PCR amplification (MASA).

OBJECTIVE: The somatic point mutation in the BRAF gene, which results in a valine-to-glutamate substitution at residue 600 (BRAF(V600E)), is an ideal hallmark of papillary thyroid carcinoma (PTC). However, its prevalence is varyingly reported in different studies, and its expression in the follicular variant PTC is controversial, reducing its potential usefulness as diagnostic marker. DESIGN AND METHODS: We developed an assay based on mutant allele-specific PCR amplification (MASA) to detect BRAF mutation. We compared the sensitivity of MASA, single-strand conformation polymorphism (SSCP) and direct DNA sequencing of PCR products. Then, we used MASA 78 to analyze 78 archival thyroid tissues, including normal samples, follicular adenomas, follicular carcinomas and PTC. RESULTS: The MASA assay proved to be a more sensitive method than SSCP and DNA sequencing of PCR products. BRAF mutation was found by MASA in 19/43 (44.2%) of PTC, including 14/31 (45.2%) classic forms and 5/12 (41.7%) follicular variants. No mutations of BRAF were detected in the normal thyroid tissues, nor in follicular adenomas or follicular carcinomas. No correlation was found between BRAF mutation and clinicopathologic features nor with recurrence during a postoperative follow-up period of 4-11 years. BRAF(V600E) significantly correlated with absence of node metastasis. CONCLUSIONS: BRAF(V600E) is present in PTC, both in the classic form and in follicular variant with similar prevalence. No correlation was found between BRAF mutation and aggressive clinical behavior. MASA-PCR proved to be a specific, sensitive and reliable method to detect BRAF T1799A in DNA extracted from different sources, including cytologic samples obtained either fresh or from archival glass slides. We propose this method as a useful tool to improve accuracy of preoperative diagnosis identifying PTC from biopsies with indeterminate cytologic findings.