Centrosome Linker-induced Tetraploid Segregation Errors Link Rhabdoid Phenotypes and Lethal Colorectal Cancers.

Centrosome anomalies contribute to tumorigenesis, but it remains unclear how they are generated in lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and BRAFV600E-mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of rootletin (CROCC), a centrosome linker component required for centrosome cohesion and separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent in a subgroup of BRAFV600E-mutant and microsatellite stable (MSS) rhabdoid colorectal cancers, but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC expression in near-diploid BRAFV600E-mutant/MSI colon cancer cells disrupts bipolar mitotic spindle architecture, promotes tetraploid segregation errors, resulting in a highly aggressive rhabdoid-like phenotype in vitro Restoring near-wild-type levels of CROCC in a metastatic model harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to centrosome spindle pole stabilizing agents in vitro These data shed light on a previously unknown link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into pathways underlying genome instability.Implications: Mis-segregation of chromosomes is a prominent feature of chromosome instability and intratumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. This study provides insight into the mechanism by which defects in rootletin, a centrosome linker component causes tetraploid segregation errors and phenotypic transition to a clinically devastating form of malignant rhabdoid tumor. Mol Cancer Res; 16(9); 1385-95. ©2018 AACR.

Insulin generates free radicals by an NAD(P)H, phosphatidylinositol 3'-kinase-dependent mechanism in human skin fibroblasts ex vivo.

Oxidative stress may be involved in the development of vascular complications associated with diabetes; however, the molecular mechanism responsible for increased production of free radicals in diabetes remains uncertain. Therefore, we examined whether acute hyperinsulinemia increases the production of free radicals and whether this condition affects proliferative extracellular signal-regulated kinase (ERK-1 and -2) signaling in human fibroblasts in vitro. Insulin treatment significantly increased intracellular superoxide anion (O(2)(-)) production, an effect completely abolished by Tiron, a cell-permeable superoxide dismutase (SOD) mimetic and by polyethylene glycol (PEG)-SOD, but not by PEG catalase. Furthermore, insulin-induced O(2)(-) production was attenuated by the NAD(P)H inhibitor apocynin, but not by rotenone or oxypurinol. Inhibition of the phosphatidylinositol 3'-kinase (PI 3'-kinase) pathway with LY294002 blocked insulin-stimulated O(2)(-) production, suggesting a direct involvement of PI 3'-kinase in the activation of NAD(P)H oxidase. The insulin-induced free radical production led to membranous translocation of p47phox and markedly enhanced ERK-1 and -2 activation in human fibroblasts. In conclusion, these findings provided direct evidence that elevated insulin levels generate O(2)(-) by an NAD(P)H-dependent mechanism that involves the activation of PI 3'-kinase and stimulates ERK-1- and ERK-2-dependent pathways. This effect of insulin may contribute to the pathogenesis and progression of cardiovascular disease in the insulin resistance syndrome.

Abnormal regulation of G protein alpha(i2) subunit in skin fibroblasts from insulin-resistant hypertensive individuals.

BACKGROUND: Studies in experimental animals and human cells have demonstrated increased intracellular calcium (Ca(i2) signalling and Galphai signal transduction associated with hypertension. We have recently shown that angiotensin II-induced mobilization of Ca(i2) is enhanced in fibroblasts from hypertensive individuals in comparison with that in normotensive individuals and that it is blunted by insulin and pertussis toxin in insulin-sensitive, but not in insulin-resistant, patients. This suggests that G(i)-mediated signal transduction is reduced in insulin-resistant hypertension. OBJECTIVE: To investigate the expression and regulation of Galpha(i2) subunit in insulin-sensitive and insulin-resistant hypertensive individuals. METHODS: G protein alpha(i2) subunit mRNA was measured in cultured skin fibroblasts from patients with insulin-sensitive and insulin-resistant hypertension, by real-time reverse transcriptase polymerase chain reaction. We also investigated the effects of short-term exposure to fetal calf serum, angiotensin II and insulin, alone and in combination, on the expression of Galpha(i2) in vitro. Spectrofluorophotometric measurement of free Cai was performed in monolayers of 24 h serum-deprived cells in basal conditions and after exposure to angiotensin II, with and without pre-incubation with insulin. RESULTS: Expression of Galpha(i2) was significantly greater in fibroblasts from hypertensive individuals than in normotensive individuals and the increase was unrelated to age and body mass. The difference was largely accounted for by greater values in insulin-sensitive than in insulin-resistant hypertensive individuals. In fibroblasts from those with insulin-sensitive hypertension, angiotensin II and insulin were additive to fetal calf serum in increasing the expression of Galpha(i2). In these patients, insulin blunted the angiotensin-II induced Cai transient. In contrast, in those with insulin-resistant hypertension, Galpha(i2) was lower and unresponsive to angiotensin II and insulin. Finally, in fibroblasts from insulin-resistant patients, insulin was unable to reduce the angiotensin II-induced Cai peak. CONCLUSIONS: A subnormal Galpha(i2)-mediated signal transduction may be involved in the pathogenesis of cellular insulin resistance in hypertension. This novel Galpha(i2)-mediated signal transduction associated with insulin sensitivity in fibroblasts may help to control excessive angiotensin II signalling.

Effects of angiotensin II and insulin on ERK1/2 activation in fibroblasts from hypertensive patients.

BACKGROUND: Insulin resistance, a frequent finding in hypertensive patients, leads to accelerated cardiovascular damage. It has been suggested that a crosstalk between angiotensin II and insulin signaling pathways may provoke insulin resistance, and may contribute to the development of cardiovascular damage. To identify a common pathophysiologic pathway between metabolic disorders and cardiovascular remodeling, we investigated the effect of angiotensin II and insulin on extracellular signal regulated kinases 1 and 2 (ERK1/2), isoforms of mitogen-activated protein kinases (MAPK) involved in cellular proliferation and extracellular matrix deposition. METHODS: Skin fibroblasts from normotensive subjects, insulin sensitive hypertensive subjects, and insulin resistant hypertensive subjects were cultured and used after four passages. The ERK1/2 expression and phosphorylation were measured by Western blot using specific antibodies, respectively anti-ERK1/2 and anti-pERK1/2. Expression of AT1 receptor for angiotensin II was determined by reverse transcriptase-polymerase chain reaction in real time. RESULTS: The ERK1/2 were similarly expressed in skin fibroblasts from all groups; ERK1/2 phosporylation evoked by angiotensin II was significantly higher in fibroblasts from hypertensive patients in comparison to normotensive subjects, but the increase was observed only in insulin resistant hypertensive subjects. The effect of insulin on ERK1/2 phosphorylation was not significantly different in the three groups. Treatment with the combination of insulin and angiotensin II increased ERK1/2 phosphorylation to a greater extent in comparison to the single agonists in normotensive subjects and in insulin sensitive but not in insulin resistant hypertensive subjects. CONCLUSIONS: Angiotensin II stimulated ERK1/2 activation is increased in insulin resistant hypertensive subjects, and it may play a role in the pathogenesis of insulin resistance and accelerated cardiovascular damage.

Regulation of glomerular filtration in essential hypertension: role of abnormal Na+ transport and atrial natriuretic peptide.

BACKGROUND: Many studies conducted in the last two decades have aroused interest in the role of glomerular hyperfiltration in the pathogenesis of renal damage in hypertension. Glomerular hyperfiltration has been mainly attributed to intraglomerular hypertension and overactivity of the renin-angiotensin system, but not much is known about the role of excessive renal proximal tubule Na+ reabsorption, which may activate tubuloglomerular feedback and increase glomerular filtration. Therefore, we evaluated the relationships between glomerular hemodynamics, plasma atrial natriuretic peptide (ANP) concentration, proximal tubule Na+ reabsorption, assessed by renal Li+ reabsorption, and sodium-lithium exchange (NLE) in circulating erythrocytes ex vivo, a marker of increased protein expression of isoform 3 of the sodium-proton exchanger (NHE-3) in the proximal tubule, in essential hypertensive patients. METHODS: 32 patients with essential hypertension were investigated after a two week placebo wash out period and after four-weeks treatment with open-label angiotensin converting enzyme inhibitors (ACEI). Before and after active treatment the following parameters were assessed: blood pressure (mercury sphygmomanometry), glomerular filtration rate (GFR), renal plasma flow, and lithium clearance (clearances of inulin, PAH and orally administered lithium, respectively), plasma ANP (radioimmunoassay), and, only at baseline, plasma renin activity, plasma and urinary aldosterone (radioimmunoassay) and NLE (sodium stimulated lithium efflux). RESULTS: Baseline GFR was positively correlated with NLE, Li+ clearance and ANP, and the patients with elevated NLE (> or =0.4 mmol/L cell/h) (31%) had higher GFR and ANP than the patients with normal NLE, but similar plasma renin activity, plasma and urinary aldosterone. ACEI reduced GFR and its change was negatively correlated with pretreatment GFR and NLE and positively with the change of proximal tubule Na+ reabsorption. After ACEI, ANP increased in patients with normal NLE but not in those with high NLE (p<0.001 for the difference). CONCLUSION: Increased proximal tubule Na+ reabsorption contributes to the pathophysiology of glomerular hyperfiltration in patients with essential hypertension, and is compensated by increased ANP levels. It can be corrected by short-term ACEI treatment.

KRAS mutations and M2PK upregulation in stool samples from individuals with positive fecal occult blood tests screened for colorectal cancer.

BACKGROUND: Screening for colorectal cancer (CRC) requires non-invasive methods of high diagnostic sensitivity and specificity. We evaluated the measurement of genetic and protein biomarkers of CRC in stool samples with the aim of testing their clinical utility in a CRC screening program. PATIENTS AND METHODS: Individuals aged 53-75 years who were at risk of CRC and immunochemical fecal occult blood test (iFOBT) positive were invited to submit stool samples for molecular testing prior to colonoscopy. KRAS codon 12 Gly→Asp, Gly, Val, and codon 13 Gly→Cys gene mutations were tested using an in-house real-time ARMS PCR method. M2PK levels in stool samples were measured utilizing a commercial ELISA kit. RESULTS: At colonoscopy, 7.6% of patients were found to have CRC, 50% had adenomas, 10.6% had hyperplastic polyps, 20.2% had diverticulosis and hemorrhoids, and 11.6% had normal mucosa. The best sensitivity for CRC (50%) was found in those cases where M2PK and KRAS abnormalities coexisted. M2PK showed a detection rate of 40.3% for adenomas but the combination of M2PK and KRAS abnormalities was found in only 5.7% of adenomas (P <0.01). iFOBT was false positive in 31.8% of cases in which colonoscopy excluded neoplastic lesions, while the coexistence of molecular and enzymatic abnormalities was more specific with false positive rates between 8.3% and 9.0% (P <0.05). CONCLUSION: Our molecular screening approach demonstrates that detection of cancer-associated biomarkers measured in iFOBT-positive stool samples could help separate true from false positives in a FOBT-based screening process. M2PK showed particular promise for the detection of CRC and adenomas.

Rhabdoid carcinoma of the colon: a distinct entity with a very aggressive behavior: a case report associated with a polyposis coli and review of the literature.

Rhabdoid colon tumors (RCTs) are rare lesions whose existence as an independent distinct entity remains controversial. To date, 6 RCTs have been reported. This study reports a novel case associated with polyposis coli in a 73-year-old woman. Histologically, the neoplasia was heterogeneous consisting of an adenocarcinoma associated with rhabdoid features. In rhabdoid component, an intense expression of MSH2 was noted but MLH1 was negative. A BRAF V600E mutation and no KRAS mutations were identified. The promoter regions of subset of genes highly specific to characterize the CIMP status (NEUROG1, IGF2, RUNX3, SOCS1, including MLH1) were hypermethylated, suggesting the presence of CIMP+ and MSI high tumor. In conclusion, all RCTs have similar clinical features. The presence of polyposis and adenocarcinoma component as well as the expression of mesenchymal marker suggests a sarcomatous dedifferentiation. It is argued that RCT could be a very aggressive entity of colon, which could benefit from new biological colonic treatments.

Comparison of JAK2V617F mutation assessment employing different molecular diagnostic techniques.

BACKGROUND: The JAK2(V617F) mutation is present in the majority of patients with polycythaemia vera and in approximately half of patients with essential thrombocythaemia and primary myelofibrosis. In this study we compare the results of JAK2(V617F) mutation detection using three different molecular techniques in the same group of patients affected by essential thrombocythaemia. PATIENTS AND METHODS: The JAK2 mutation was investigated with a qualitative method in 115 consecutive outpatients with a diagnosis of essential thrombocythaemia made according to WHO 2001 criteria. In 48/115 (41.7%) the allele burden was also evaluated with two different qualitative methods, of which one was a method developed in-house and the other was a commercially available method. RESULTS: The JAK2(V617F) mutation was detected by the qualitative method in 81/115 (69.6%) of the patients. Among the 48/115 patients in whom all three methods were applied, the qualitative method detected the mutation in 38 (79%). According to the quantitative method developed in-house, the mutation was present in 35/48 (73%) of the patients: of these, 2/35 (5.7%) patients were homozygous for the JAK2(V617F) mutation. The commercial quantitative method showed the mutation in 37/48 (77%) patients: of these, 9/37 (18%) patients were homozygous. Three of the 13 patients in whom no mutation was detected by the in-house method were positive for the JAK2(V617F) according to the commercial method. In one patient the search for the JAK2(V617F) mutation was positive with the in-house method but negative with the commercial kit. CONCLUSION: Detection of the JAK2(V617F) mutation may depend on the molecular technique used. Considering that detection of this mutation will not only have a diagnostic value, but also a role in treatment given the development of JAK2(V617F) pathway inhibiting drugs, indications on a reference molecular diagnostic technique for JAK2(V617F) assessment and quantification of its allele burden from a panel of experts are warranted.

Different effect of ouabain on endothelin-1-induced extracellular signal-regulated kinase stimulation in rat heart and tail artery.

Endogenous ouabain may play a role in the control of cardiovascular system function. In this study, we investigated the effects of a long-term ouabain treatment on basal and endothelin-1 (ET-1)-induced phosphorylation of cardiac and vascular extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2), which are involved in several cardiac and vascular physiologic and pathologic conditions. Our results show that the hearts from ouabain-treated rats have a higher basal level of ERK-1 and ERK-2 phosphorylation compared with untreated rats. Perfusion of the hearts with ET-1 increased ERK-1 and ERK-2 phosphorylation both in ouabain-treated and in control rats, with a larger stimulatory effect in ouabain-treated animals. On the contrary, exposure of endothelium-free tail artery to ET-1 increased ERKs phosphorylation both in treated and untreated rats, but this effect was blunted in ouabain-treated rats. These findings demonstrate that ouabain treatment has opposite effects on basal and ET-1-induced ERKs phosphorylation in the heart and in the tail artery of the rat.