Luminal breast cancer metastases and tumor arousal from dormancy are promoted by direct actions of estradiol and progesterone on the malignant cells.

INTRODUCTION: Luminal, estrogen receptor-positive (ER(+)) breast cancers can metastasize but lie dormant for years before recurrences prove lethal. Understanding the roles of estrogen (E) or progestin (P) in development of luminal metastases or in arousal from dormancy is hindered by few preclinical models. We have developed such models. METHODS: Immunocompromised, ovariectomized (ovx'd) mice were intracardiac-injected with luminal or basal human breast cancer cells. Four lines were tested: luminal ER(+)PR(+) cytokeratin 5-negative (CK5(-)) E3 and MCF-7 cells, basal ER(-)PR(-)CK5(+) estrogen withdrawn-line 8 (EWD8) cells, and basal ER(-)PR(-)CK5(-) MDA-MB-231 cells. Development of micrometastases or macrometastases was quantified in ovx'd mice and in mice supplemented with E or P or both. Metastatic deposits were analyzed by immunohistochemistry for luminal, basal, and proliferation markers. RESULTS: ER(-)PR(-) cells generated macrometastases in multiple organs in the absence or presence of hormones. By contrast, ovx'd mice injected with ER(+)PR(+) cells appeared to be metastases-free until they were supplemented with E or E+P. Furthermore, unlike parental ER(+)PR(+)CK5(-) cells, luminal metastases were heterogeneous, containing a significant (6% to 30%) proportion of non-proliferative ER(-)PR(-)CK5(+) cells that would be chemotherapy-resistant. Additionally, because these cells lack receptors, they would also be endocrine therapy-resistant. With regard to ovx'd control mice injected with ER(+)PR(+) cells that appeared to be metastases-free, systematic pathologic analysis of organs showed that some harbor a reservoir of dormant micrometastases that are ER(+) but PR(-). Such cells may also be endocrine therapy- and chemotherapy-resistant. Their emergence as macrometastases can be triggered by E or E+P restoration. CONCLUSIONS: We conclude that hormones promote development of multi-organ macrometastases in luminal disease. The metastases display a disturbing heterogeneity, containing newly emergent ER(-)PR(-) subpopulations that would be resistant to endocrine therapy and chemotherapy. Similar cells are found in luminal metastases of patients. Furthermore, lack of hormones is not protective. While no overt metastases form in ovx'd mice, luminal tumor cells can seed distant organs, where they remain dormant as micrometastases and sheltered from therapies but arousable by hormone repletion. This has implications for breast cancer survivors or women with occult disease who are prescribed hormones for contraception or replacement purposes.

MAP kinase kinase kinase-2 (MEKK2) regulates hypertrophic remodeling of the right ventricle in hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) results in pressure overload of the right ventricle (RV) of the heart, initiating pathological RV remodeling and ultimately leading to right heart failure. Substantial research indicates that signaling through the MAPK superfamily mediates pathological cardiac remodeling. These considerations led us to test the hypothesis that the regulatory protein MAPKKK-2 (MEKK2) contributes to RV hypertrophy in hypoxia-induced PH. Transgenic mice with global knockout of MEKK2 (MEKK2(-/-) mice) and age-matched wild-type (WT) mice were exposed to chronic hypobaric hypoxia (10% O(2), 6 wk) and compared with animals under normoxia. Exposure to chronic hypoxia induced PH in WT and MEKK2(-/-) mice. In response to PH, WT mice showed RV hypertrophy, demonstrated as increased ratio of RV weight to body weight, increased RV wall thickness at diastole, and increased cardiac myocyte size compared with normoxic control animals. In contrast, each of these measures of RV hypertrophy seen in WT mice after chronic hypoxia was attenuated in MEKK2(-/-) mice. Furthermore, chronic hypoxia elicited altered programs of hypertrophic and inflammatory gene expression consistent with pathological RV remodeling in WT mice; MEKK2 deletion selectively inhibited inflammatory gene expression compared with WT mice. The actions of MEKK2 were mediated in part through regulation of the abundance and phosphorylation of its effector, ERK5. In conclusion, signaling by MEKK2 contributes to RV hypertrophy and altered myocardial inflammatory gene expression in response to hypoxia-induced PH. Therapies targeting MEKK2 may protect the myocardium from hypertrophy and pathological remodeling in human PH.

Brief Report: Case Comparison of Therapy With the Histone Deacetylase Inhibitor Vorinostat in a Neonatal Calf Model of Pulmonary Hypertension.

Pulmonary hypertension (PH) is an incurable condition in humans; driven by pulmonary vascular remodeling partially mediated by epigenetic mechanisms; and leading to right ventricular hypertrophy, failure, and death. We hypothesized that targeting chromatin-modifying histone deacetylases may provide benefit. In this Brief Report we describe case comparison studies using the histone deacetylase inhibitor vorinostat (suberanilohydroxamic acid, 5 mg/kg/day for the first 5 study days) in an established model of severe neonatal bovine PH induced by 14 days of environmental hypoxia. Echocardiographic, hemodynamic, and pharmacokinetic data were obtained in hypoxia-exposed (one each, vorinostat-treated vs. untreated) and normoxic vorinostat-treated control animals (n = 2). Echocardiography detected PH changes by day 4 and severe PH over 14 days of continued hypoxic exposure. RV dysfunction at day 4 was less severe in vorinostat-treated compared to untreated hypoxic calves. Cardioprotective effects were partially maintained following cessation of treatment through the duration of hypoxic exposure, accompanied by hemodynamic evidence suggestive of reduced pulmonary vascular stiffening, and modulated expression of HDAC1 protein and genes involved in RV and pulmonary vascular remodeling and pathological RV hypertrophy. Control calves did not develop PH, nor show adverse cardiac or clinical effects. These results provide novel translation of epigenetic-directed therapy to a large animal severe PH model that recapitulates important features of human disease.

Stage-specific changes in myofilament protein phosphorylation following myocardial infarction in mice.

The response of cardiac muscle to an insult such as myocardial infarction includes changes in the expression of numerous signaling proteins and modulation of gene expression, as well as post-translational modifications of existing proteins. Most studies to date have defined these in end-stage cardiac muscle thus obviating consideration of the temporal progression that causes the heart to transition from a compensated to a decompensated phenotype. To explore these transitions, we examined contractile protein biochemistry in a mouse MI model at two early time points: 2 days and 2 weeks post-infarct and at two later time points: 2 and 4 months post-infarct. Phosphorylation of myofilament proteins was analyzed using phosphospecific staining of polyacrylamide gels, and whenever possible, phosphospecific antibodies. Phosphorylation of myosin binding protein c, the myosin regulatory light chain and troponin I were all decreased relative to sham operated animals at both early time points. However, by 2 months, total phosphorylation of all the major myofilament proteins normalized and at both 2 and 4 months, there was a significant increase in troponin I phosphorylation. One-dimensional IEF of troponin I coupled with phospho-specific antibody analysis demonstrated a redistribution of phosphorylation sites with a significant initial decline at the putative PKA sites, Serine 22,23, and a subsequent increase at the putative PKC site, serine 43,45. These data suggest that temporal changes in myofilament protein phosphorylation contribute both to the initial compensatory hyperdynamic response to myocardial infarction and subsequently to the gradual progression to myocardial failure.

Prolonged administration of a dithiol antioxidant protects against ventricular remodeling due to ischemia-reperfusion in mice.

The prolonged production of reactive oxygen species due to ischemia-reperfusion (I/R) is a potential cause of the pathological remodeling that frequently precedes heart failure. We tested the ability of a potent dithiol antioxidant, bucillamine, to protect against the long-term consequences of I/R injury in a murine model of myocardial infarction. After transiently occluding the left anterior descending coronary artery for 30 min, saline or bucillamine (10 microg/g body wt) was injected intravenously as a bolus within the first 5 min of reperfusion. The antioxidant treatment continued with daily subcutaneous injections for 4 wk. There were no differences in infarct sizes between bucillamine- and saline-treated animals. After 4 wk of reperfusion, cardiac hypertrophy was decreased by bucillamine treatment (ventricular weight-to-body weight ratios: I/R + saline, 4.5 +/- 0.2 mg/g vs. I/R + bucillamine, 4.2 +/- 0.1 mg/g; means +/- SE; P < 0.05). Additionally, the hearts of bucillamine-treated mice had improved contractile function (echocardiographic measurement of fractional shortening) relative to saline controls: I/R + saline, 32 +/- 3%, versus I/R + bucillamine, 41 +/- 4% (P < 0.05). Finally, I/R-induced injury in the saline-treated mice was accompanied by a fetal pattern of gene expression determined by ribonuclease protection assay that was consistent with pathological cardiac hypertrophy and remodeling [increased atrial natriuretic peptide, beta-myosin heavy chain (MHC), skeletal alpha-actin; decreased sarco(endo)plasmic reticulum Ca2+ ATPase 2a, and alpha-MHC-to-beta-MHC ratio]. These changes in gene expression were significantly attenuated by bucillamine. Therefore, treatment with a dithiol antioxidant for 4 wk after I/R preserved ventricular function and prevented the abnormal pattern of gene expression associated with pathological cardiac remodeling.

The cardiac fibroblast: therapeutic target in myocardial remodeling and failure.

Cardiac fibroblasts play a central role in the maintenance of extracellular matrix in the normal heart and as mediators of inflammatory and fibrotic myocardial remodeling in the injured and failing heart. In this review, we evaluate the cardiac fibroblast as a therapeutic target in heart disease. Unique features of cardiac fibroblast cell biology are discussed in relation to normal and pathophysiological cardiac function. The contribution of cardiac fibrosis as an independent risk factor in the outcome of heart failure is considered. Candidate drug therapies that derive benefit from actions on cardiac fibroblasts are summarized, including inhibitors of angiotensin-aldosterone systems, endothelin receptor antagonists, statins, anticytokine therapies, matrix metalloproteinase inhibitors, and novel antifibrotic/anti-inflammatory agents. These findings point the way to future challenges in cardiac fibroblast biology and pharmacotherapy.