The anti-HER3 antibody patritumab abrogates cetuximab resistance mediated by heregulin in colorectal cancer cells.

We previously showed that tumor-derived heregulin, a ligand for HER3, is associated with both de novo and acquired resistance to cetuximab. We have now examined whether patritumab, a novel neutralizing monoclonal antibody to HER3, is able to overcome such resistance. Human colorectal cancer (DiFi) cells that are highly sensitive to cetuximab were engineered to stably express heregulin by retroviral infection, and the effects of cetuximab and patritumab on the resulting DiFi-HRG cells were examined. DiFi-HRG cells released substantial amounts of heregulin and showed resistance to cetuximab. Cetuximab alone inhibited EGFR and ERK phosphorylation in DiFi-HRG cells, but it had no effect on the phosphorylation of HER2, HER3, or AKT, suggesting that sustained AKT activation by HER2 and HER3 underlies cetuximab resistance in these cells. In contrast, patritumab in combination with cetuximab markedly inhibited the phosphorylation of EGFR, HER2, HER3, ERK, and AKT. The combination therapy also inhibited the growth of DiFi-HRG tumor xenografts in nude mice to a greater extent than did treatment with either drug alone. Activation of HER2-HER3 signaling associated with the operation of a heregulin autocrine loop confers resistance to cetuximab, and patritumab is able to restore cetuximab sensitivity through inhibition of heregulin-induced HER3 activation.

Tyrosine phosphoproteomics identifies both codrivers and cotargeting strategies for T790M-related EGFR-TKI resistance in non-small cell lung cancer.

PURPOSE: Irreversible EGFR-tyrosine kinase inhibitors (TKI) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gatekeeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. EXPERIMENTAL DESIGN: We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined. RESULTS: We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib-treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced antitumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M-mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. CONCLUSIONS: Our results identified both codrivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in patients with NSCLC with acquired T790M.

Nitric oxide synthases and heart failure - lessons from genetically manipulated mice.

Nitric oxide (NO) is synthesized by three distinct NO synthase (NOS) isoforms (neuronal, inducible, and endothelial NOS), all of which are expressed in the human heart. The roles of NOSs in the pathogenesis of heart failure have been described in pharmacological studies with NOS inhibitors. Recently, genetically engineered animals have been used. We have generated mice in which all 3 NOS isoforms are completely disrupted (triple n/i/eNOS(-/-) mice). Morphological, echocardiographic, and hemodynamic analysis were performed in wild-type, singly nNOS(-/-), iNOS(-/-), eNOS(-/-), and triple n/i/eNOS(-/-) mice. Importantly, significant left ventricular (LV) hypertrophy and diastolic dysfunction was noted only in n/i/eNOS(-/-) mice, and those pathology was similar to diastolic heart failure in humans. Finally, treatment with an angiotensin II type 1 (AT1) receptor blocker, significantly prevented those abnormalities. These results provide the evidence that AT1 receptor pathway plays a center role in the pathogenesis of cardiac disorders in the n/i/eNOS(-/-) mice. Our studies with triple n/i/eNOS(-/-) mice provide pivotal insights into an understanding of the pathophysiology of NOSs in human heart failure.

Spontaneous development of left ventricular hypertrophy and diastolic dysfunction in mice lacking all nitric oxide synthases.

BACKGROUND: The role of the nitric oxide synthase (NOS) system in cardiac architecture and function remains unknown. This point was addressed in mice that lack all 3 NOS genes. METHODS AND RESULTS: Morphological, echocardiographic, and hemodynamic analyses were performed in wild-type (WT), singly nNOS(-/-), iNOS(-/-), eNOS(-/-), and triply n/i/eNOS(-/-) mice. At 5 months of age, but not at 2 months of age, significant left ventricular (LV) hypertrophy was noted in n/i/eNOS(-/-) mice and to a lesser extent in eNOS(-/-) mice, but not in nNOS(-/-) or iNOS(-/-) mice, compared with WT mice. Importantly, significant LV diastolic dysfunction (as evaluated by echocardiographic E/A wave ratio and hemodynamic -dP/dt and Tau), with preserved LV systolic function (as assessed by echocardiographic fractional shortening and hemodynamic +dP/dt), was noted only in n/i/eNOS(-/-) mice, and this was associated with enhanced LV end-diastolic pressure and increased lung wet weight, all of which are characteristics consistent with diastolic heart failure in humans. Finally, long-term oral treatment with an angiotensin II type 1 (AT(1)) receptor blocker, olmesartan, significantly prevented all these abnormalities of n/i/eNOS(-/-) mice. CONCLUSIONS: These results provide the first direct evidence that the complete disruption of all NOSs results in LV hypertrophy and diastolic dysfunction in mice in vivo through the AT(1) receptor pathway, demonstrating a pivotal role of the endogenous NOS system in maintaining cardiac homeostasis.

Severe dyslipidaemia, atherosclerosis, and sudden cardiac death in mice lacking all NO synthases fed a high-fat diet.

AIMS: The precise role of the nitric oxide synthase (NOS) system in lipid metabolism remains to be elucidated. We addressed this point in mice that we have recently developed and that lack all three NOS isoforms. METHODS AND RESULTS: Wild-type (WT), singly, doubly, and triply NOS(-/-) mice were fed either a regular or high-cholesterol diet for 3-5 months. The high-cholesterol diet significantly increased serum low-density lipoprotein (LDL) cholesterol levels in all the genotypes when compared with the regular diet. Importantly, when compared with the WT genotype, the serum LDL cholesterol levels in the high-cholesterol diet were significantly and markedly elevated only in the triply NOS(-/-) genotype, but not in any singly or doubly NOS(-/-) genotypes, and this was associated with remarkable atherosclerosis and sudden cardiac death, which occurred mainly in the 4-5 months after the high-cholesterol diet. Finally, hepatic LDL receptor expression was markedly reduced only in the triply NOS(-/-) genotype, accounting for the diet-induced dyslipidaemia in the genotype. CONCLUSION: These results provide the first direct evidence that complete disruption of all NOS genes causes severe dyslipidaemia, atherosclerosis, and sudden cardiac death in response to a high-fat diet in mice in vivo through the down-regulation of the hepatic LDL receptor, demonstrating the critical role of the whole endogenous NOS system in maintaining lipid homeostasis.

Spontaneous myocardial infarction in mice lacking all nitric oxide synthase isoforms.

BACKGROUND: The roles of nitric oxide (NO) in the cardiovascular system have been investigated extensively in pharmacological studies with NO synthase (NOS) inhibitors and in studies with NOS isoform-deficient mice. However, because of the nonspecificity of the NOS inhibitors and the compensatory interactions among NOS isoforms (nNOS, iNOS, and eNOS), the ultimate roles of endogenous NO derived from the entire NOS system are still poorly understood. In this study, we examined this point in mice deficient in all 3 NOS isoforms (triply n/i/eNOS(-/-) mice) that we have recently developed. METHODS AND RESULTS: The triply n/i/eNOS(-/-) mice, but not singly eNOS(-/-) mice, exhibited markedly reduced survival, possibly due to spontaneous myocardial infarction accompanied by severe coronary arteriosclerotic lesions. Furthermore, the triply n/i/eNOS(-/-) mice manifested phenotypes that resembled metabolic syndrome in humans, including visceral obesity, hypertension, hypertriglyceridemia, and impaired glucose tolerance. Importantly, activation of the renin-angiotensin system was noted in the triply n/i/eNOS(-/-) mice, and long-term oral treatment with an angiotensin II type 1 receptor blocker significantly suppressed coronary arteriosclerotic lesion formation and the occurrence of spontaneous myocardial infarction and improved the prognosis of those mice, along with ameliorating the metabolic abnormalities. CONCLUSIONS: These results provide the first direct evidence that genetic disruption of the whole NOS system causes spontaneous myocardial infarction associated with multiple cardiovascular risk factors of metabolic origin in mice in vivo through the angiotensin II type 1 receptor pathway, demonstrating the critical role of the endogenous NOS system in maintaining cardiovascular and metabolic homeostasis.

Angiotensin receptor blocker improves coronary flow velocity reserve in hypertensive patients: comparison with calcium channel blocker.

Large-scale clinical studies have indicated that angiotensin receptor blockers (ARBs) have beneficial effects against cardiovascular diseases. We designed this study to compare the effects of an ARB and a calcium channel blocker (CCB) on coronary flow velocity reserve (CFVR), a predictor of cardiovascular events, as estimated using transthoracic Doppler echocardiography. Sixteen hypertensive patients (63.1+/-9.6 years old; 10 males) were randomly allocated in a double-blind fashion to valsartan (n=8, 40-80 mg/day) or nifedipine (n=8, 20-40 mg/day) groups. Age- and gender-matched subjects without hypertension were enrolled as a control group (n=12). CFVR was calculated by dividing the adenosine triphosphate-induced hyperemic flow velocity by the basal flow velocity in the left anterior descending coronary artery. Baseline characteristics and reduction in systolic and diastolic blood pressure after 6 months were similar in both groups. CFVR in the valsartan group increased from 2.34+/-0.38 to 3.10+/-0.84 at 2 months (p<0.05), and to 3.04+/-1.09 at 6 months (p<0.01). Both values became comparable to that in the control group (2.81+/-0.60). CFVR in the valsartan group was significantly higher (p<0.001) than that in the nifedipine group, which was little changed at 6 months. This discrepancy was derived from the significant increase of hyperemic velocity in the valsartan group, from 36.6+/-17.3 cm/s to 41.1+/-12.7 cm/s at 2 months, and to 48.1+/-20.2 cm/s at 6 months. We concluded that the ARB valsartan not only reduced high blood pressure but improved CFVR in hypertensive patients. However, these effects were not seen with the CCB nifedipine.