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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.

Supplementary Table 1. Clinicopathological, immunohistochemical and molecular features of the 12 rhabdoid colorectal cancers7 Supplementary Table 2. Somatic mutations of candidate genes (15 out of 20 are reported) shared by the two rhabdoid cancers at exome sequencing Supplementary Table 3. Rhabdoid colorectal cancers (RC): molecular alterations, centrosome aberrations and ploidy Supplementary Table 4. Rhabdoid tumours of infants (RI): molecular alterations, centrosome aberrations, and ploidy Supplementary Table 5: Clinicopathologic and molecular features of classical colorectal cancers analyzed Supplementary Table 6. Sequencing and Reverse transcription PCR primers Supplementary Table 6B. SMARCB1 regions covered by the custom panel used for next-generation target sequencing Supplementary Table 7. CROCC regions covered by the custom panel used for next-generation targeted sequencing Supplementary Table 8. Antibodies employed and working conditions for immunohistochemistry, immunofluorescence or western blot analysis of tissues and cell lines Supplementary Table 9. Rhabdoid colorectal cancer reported in literature between 1993-2015

The file contains supplementary Materials and Methods

S1. Whole exome sequencing analysis of two rhabdoid colorectal cancers (RC). S2. Non-silent somatic mutations in rhabdoid tumours are enriched in centrosome-microtubule components. S3. Effect of CROCC mutations on centrosome phenotype. S4. CROCC expression profiling in classical colorectal cancers (CRCs). S5. Allelic loss at the 1p36.13 locus correlates with chromosomal instability in colorectal cancer cells. S6. Centrosome and chromosome instability in colorectal cancer cells. S7. Mitotic errors and impaired cell cycle caused by stable CROCC depletion in RKO cells. S8. Invasive behavior of colorectal cancer cells triggered by CROCC depletion. S9. Mitotic aberrations suppressed by CROCC or microtubule stabilizing agents in metastatic T84 and HT29 colorectal cancer cells.

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 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.

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 AT 1 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.

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.

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.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.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.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.

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.

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 (O2−) 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 O2− 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 O2− 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 O2− 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.

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.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.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.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.

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.

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.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).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 (p0.001 for the difference).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.

Background Adrenal zona glomerulosa cells are provided with adrenomedullin receptors. Adrenomedullin has recently been found to enhance proliferation of cultured rat vascular smooth muscle cells and zona glomerulosa cells. Objective To investigate whether adrenomedullin affects rat zona glomerulosa proliferative activity through the tyrosine kinase and extracellular signal regulated kinases (ERKs) pathways. Methods Dispersed rat zona glomerulosa cells were cultured in vitro for 24 h and then exposed to adrenomedullin (10 7 mol/l), alone or in the presence of tyrphostin-23 (10 5 mol/l) or PD-98059 (10 4 mol/l), for 24 or 48 h. To assess the rate of DNA synthesis, 5-bromo-2'-deoxyuridine (BrdU, 20 mg/ml) was also added to the medium and BrdU-positive cells were detected by immunocytochemistry. The expression of ERKs and the effect of adrenomedullin on ERKs phosphorylation and activity were assayed in dispersed zona glomerulosa cells. Results Adrenomedullin significantly increased the percentage of BrdU-positive (phase-S) zona glomerulosa cells; this effect was blocked by either the tyrosine kinase inhibitor, tyrphostin-23, or the mitogen-activated protein kinase kinase (MEK-1) inhibitor, PD-98059. Both zona glomerulosa and zona fasciculata/reticularis express ERK-1 (44 kDa) and ERK-2 (42 kDa) isoforms. However, adrenomedullin phosphorylated ERK-1 and ERK-2 only in the zona glomerulosa; this effect was blunted by the MEK-1 inhibitor, PD98059, and by the calcitonin gene-related peptide type 1 (CGRP-1) receptor antagonist, CGRP 8-37 , but not by the adrenomedullin C-terminal fragment, ADM 22-52 . Conclusion Adrenomedullin stimulates the growth of rat zona glomerulosa cells through activation of CGRP-1 receptor, linked to the tyrosine kinase-MEK-1 -ERKs signalling pathway. These results confirm the complex role played by this peptide in the regulation of zona glomerulosa cell physiology.

P161 Aim: To investigate the in vivo effect of adrenomedullin (ADM) on the proliferative activity of the rat adrenal cortex, using an intact left gland in situ perfusion model. Adrenal glands were exposed to ADM and other chemicals, added to the perfusion medium, for 180 min. Results: ADM concentration-dependently increased the mitotic index and [ 3 H]thymidine incorporation into DNA in the ZG (maximally effective [ADM] being 10 -8 M), but not in inner adrenocortical layers. Both the calcitonin gene-related peptide (CGRP) type 1receptor antagonist CGRP(8-37) and the ADM C terminal fragment ADM(22-52) equipotently counteracted the effect of 10 -8 M ADM. The adenylate cyclase (AC) inhibitor SQ-22536 (10 -4 M), and the protein kinase (PK)A inhibitor H-89 (10 -5 M) did not affect ADM-elicited increase in DNA synthesis, although counteracting the ZG proliferogenic action of 10 -9 M ACTH. The phospholipase (PL) C inhibitor U-73122 (10 -5 M), and the PKC inhibitor calphostin-C (10 -5 M), which significantly inhibited the ZG proliferogenic effect of 10 -9 M angiotensin-II (Ang II) were also uneffective on ADM response. Both ADM and Ang II (10 -7 M) activated MAPK kinase (by 30 and 70%) and the phosphorylation of ERK-1 and ERK-2 kinase (by 30% and by 40%), respectively. Activation of ERK-1 and ERK-2 occurred via a MEK kinase-dependent mechanism, since it was abolished by the MEK inhibitor PD-98059. No such effects of ADM were seen in cells from inner adrenocortical layers. The growth-promoting effect of ADM was unaffected by the PLA 2 inhibitor AACOCF3 (10 -5 M), the COX inhibitor indomethacin (10 -5 M) and the mixed COX/lipoxygenase (LOX) inhibitor phenidone (10 -5 M). In contrast, it was abolished by either the tyrosine kinase (TK) inhibitor tyrphostin-23 (10 -5 M) or by PD-98059 (10 4 M). Conclusions: 1) ADM exerts a mitogenic effect in the rat ZG, through ADM(22-52)-sensitive CGRP1 receptors; 2) this effect involves activation of the TKMAPK cascade, but not of the AC/PKA-, PLC/PKC-, and COX- or LOX-dependent signaling pathways.

Glycemic spikes may negatively affect the long-term prognosis of patients with diabetes. Extracellular signal-regulated kinases (ERKs) are intracellular mediators of cell proliferation, and they can be activated in response to high glucose levels. However, the modifications of their activity in response to hyperglycemia have been poorly investigated, in vivo, in humans. Thus, we sought to determine in circulating monocytes: 1) the role of hyperglycemia in ERKs activity and phosphorylation, and 2) whether hyperglycemia affects mitogen-activated protein kinase kinase (MEK) activity and mitogen-activated protein phosphatase-1 (MKP-1) expression. These goals were performed in five normal subjects. Baseline monocyte ERKs activity was 60 ± 5 pmol/min·mg protein; when exogenous hyperglycemia was induced, both monocyte ERKs activity (81 ± 11 pmol/min·mg protein; P < 0.05) and phosphorylation significantly increased (P < 0.01). MEK activity was significantly increased by hyperglycemia (1251 ± 136 vs. 2000 ± 42 cpm; P = 0.0017), whereas no changes were observed in MKP-1 expression. We conclude that hyperglycemia acutely stimulates ERKs activity and phosphorylation in human monocytes by the MEK pathway in vivo. These findings may be relevant in understanding the negative role of acute hyperglycemia on monocyte pathophysiology.

Abstract —We have recently shown that insulin attenuates angiotensin II–induced intracellular Ca 2+ mobilization in human skin fibroblasts from normotensive subjects. This study was designed to investigate the effects of angiotensin II and the interactions between insulin and angiotensin II on intracellular Ca 2+ mobilization in skin fibroblasts from patients with essential hypertension. Fibroblasts were obtained from 9 normotensives and 18 hypertensives. Spectrofluorophotometric free Ca 2+ measurement was performed in monolayers of 24-hour serum-deprived cells. Resting intracellular Ca 2+ level and angiotensin II–stimulated intracellular Ca 2+ peak were higher in fibroblasts from hypertensives compared with those from normotensives. The effect of acute insulin exposure was evaluated in fibroblasts from hypertensives subdivided on the basis of insulin sensitivity. In insulin-sensitive hypertensives, insulin significantly blunted the effects of angiotensin II on intracellular Ca 2+ response, whereas in insulin-resistant patients, insulin did not modify intracellular Ca 2+ response to angiotensin II. Pertussis toxin, a G iα -inhibitor, reduced angiotensin II–stimulated Ca 2+ peak in insulin-sensitive but not in insulin-resistant hypertensives. In conclusion, the effects of angiotensin II on intracellular Ca 2+ mobilization are more pronounced in fibroblasts from hypertensives compared with those from normotensives, and the inhibitory effect of insulin is blunted in insulin-resistant hypertensives by a G iα pertussis toxin–sensitive abnormality.