No abstract

Breast cancers frequently progress or relapse during targeted therapy, but the molecular mechanisms that enable escape remain poorly understood. We elucidated genetic determinants underlying tumor escape in a transgenic mouse model of Wnt pathway-driven breast cancer, wherein targeted therapy is simulated by abrogating doxycycline-dependent Wnt1 transgene expression within established tumors. In mice with intact tumor suppressor pathways, tumors typically circumvented doxycycline withdrawal by reactivating Wnt signaling, either via aberrant (doxycycline-independent) Wnt1 transgene expression or via acquired somatic mutations in the gene encoding β-catenin. Germline introduction of mutant tumor suppressor alleles into the model altered the timing and mode of tumor escape. Relapses occurring in the context of null Ink4a/Arf alleles (disrupting both the p16 Ink4a and p19 Arf tumor suppressors) arose quickly and rarely reactivated the Wnt pathway. In addition, Ink4a/Arf-deficient relapses resembled p53-deficient relapses in that both displayed morphologic and molecular hallmarks of an epithelial-to-mesenchymal transition (EMT). Notably, Ink4a/Arf deficiency promoted relapse in the absence of gross genomic instability. Moreover, Ink4a/Arf-encoded proteins differed in their capacity to suppress oncogene independence. Isolated p19 Arf deficiency mirrored p53 deficiency in that both promoted rapid, EMT-associated mammary tumor escape, whereas isolated p16 Ink4a deficiency failed to accelerate relapse. Thus, p19 Arf /p53 pathway lesions may promote mammary cancer relapse even when inhibition of a targeted oncogenic signaling pathway remains in force. IntroductionBreast cancer research offers a clinically important venue for exploring resistance to targeted therapy. Antagonists of estrogen receptor-dependent (ER-dependent) and human epidermal growth factor receptor 2 (HER2-dependent) signaling are mainstays of modern breast cancer treatment that enhance cure rates when applied against early-stage disease and contribute to disease remissions when applied against late-stage disease (1, 2). Even so, potent targeted agents impose strong selective pressure that ultimately favors tumor escape, wherein treatment-resistant cancer cells survive and proliferate (3). Indeed, resistance to targeted agents, when not encountered de novo, routinely emerges during treatment (4, 5). As a result, targeted agents supplement traditional breast cancer treatment strategies but do not yet obviate the need for surgery, radiation, and cytotoxic chemotherapy. Moreover, incorporating targeted agents into routine clinical practice does not yet permit cure of advanced disease. Thus, tumor escape sets profound limits on the clinical usefulness of targeted therapy in breast cancer patients.In principle, tumors can escape growth constraints imposed by targeted therapy either by reactivating the targeted signaling pathway or by perturbing untargeted compensatory pathways. Both mechanisms appear capable of promoting tumor escape in breast cancer patients....

Glucuronidation, mediated by UDP-glucuronosyltransferases (UGTs), affects the actions and disposition of diverse endo-and xenobiotics. In the case of catecholestrogens (CEs), glucuronidation is likely to block their oxidation to quinone estrogens that are the putative mediators of CEs' actions as initiators of cancers. The goal of this study was to determine whether UGT2B7, the isoenzyme with a high affinity for 4-hydroxyestrone, is expressed in human breast parenchyma. Glucuronidation of 4-hydroxyestrone has relevance to breast carcinogenesis because quinone metabolites of 4-hydroxylated CEs can form potentially mutagenic depurinating DNA adducts, and because in breast tissue estrone is likely to be the predominant estrogen available for 4-hydroxylation. Using reverse transcriptase-polymerase chain reaction, immunocytochemistry, immunoblot analyses, and assays of glucuronidation of 4-hydroxyestrone, we show that UGT2B7 is expressed in human mammary epithelium, and that its expression is dramatically reduced in invasive breast cancers. In many in situ carcinomas, however, 4-hydroxyestrone immunostaining was not only preserved but even more intense than in normal mammary epithelium. The finding of reduced UGT2B7 protein and glucuronidation of 4-hydroxyestrone in invasive cancers suggests a tumor-suppressor function for the enzyme. Recent identification of all-trans retinoic acid as a substrate of UGT2B7 suggests that this function includes the generation of retinoyl-␤-glucuronide, a potent mediator of actions of retinoids important for maintaining epithelia in a differentiated state. Current knowledge does not provide any ready explanation for the apparent increase in UGT2B7 expression in carcinomas in situ. However, this finding, together with reduced immunostaining at loci showing breach of the basement membrane (microinvasion), suggests involvement of UGT2B7-catalyzed reaction(s) in protection against invasion of surrounding tissue by cancer cells.

N G-nitro-l-arginine methyl ester (l-NAME; 250 μg/5 μl), an inhibitor of NO synthase, or the vehicle artificial cerebrospinal fluid (aCSF; 5 μl) was administered intracerebroventricularly to conscious rats hemorrhaged (0.7 ml/min) to a 20% volume depletion. Hypotension was maximal 5 min after hemorrhage ended, with compensatory recovery to basal levels 20 min later, regardless of drug treatment.l-NAME, however, elevated ( P < 0.05) blood pressure (vs. aCSF controls) 40–45 min after intracerebroventricular administration. In normovolemic rats, l-NAME produced a significant pressor response and increased plasma levels of vasopressin (VP) and oxytocin (OT). After hemorrhage, both hormone levels increased, but only OT was further enhanced byl-NAME. Thus centrally produced NO tonically inhibits OT and VP secretion under basal normovolemic conditions and selectively inhibits OT release during hypovolemia. Hemorrhage increased the rates of glucose utilization in the neural lobe, indicative of enhanced efferent neural functional activity.l-NAME further enhanced the metabolic activity in the entire hypothalamoneurohypophysial system of hemorrhaged animals. Several other brain structures involved in the regulation of blood pressure and the stress response were also metabolically affected by the hemorrhage andl-NAME.

NG-nitro-L-arginine methyl ester (L-NAME, 250 micrograms/5 microliters), an inhibitor of nitric oxide (NO) synthase, or artificial cerebrospinal fluid (5 microliters) was administered intracerebroventricularly to conscious naive rats or to rats treated subcutaneously (15 microliters/kg) with NaCl (0.15, 0.45, or 1.0 M) or given a needle prick only. Intracerebroventricular injection of L-NAME increased plasma concentration of vasopressin (VP) and oxytocin (OT) in control naive rats, indicating that NO tonically inhibits basal secretion of both hormones during isosmotic isovolemic conditions. Osmotic stimulation with hypertonic saline (0.45 and 1.0 M NaCl) elevated plasma levels of both hormones as expected. Central blockade of NO synthase further enhanced secretion of OT during mild, but not strong, osmotic stimulation, whereas the high levels of VP remained unaffected by L-NAME. In animals treated with the needle prick or 0.15 M NaCl, only OT levels were increased after L-NAME. Therefore, NO selectively inhibits OT release in response to a painful stimulus (needle prick) and moderate osmotic stimulation to promote a preferential release of VP. A transient pressor response was observed after subcutaneous injection of 0.15 and 0.45 M NaCl, but a sustained response was obtained after 1.0 M NaCl. Regardless of whether the animals received NaCl solutions, however, treatment with L-NAME elevated blood pressure in all animals. Thus NO-induced vasodilation maintains basal arterial blood pressure and limits the pressor response to osmotic stimulation.

Catechol-O-methyltransferase (COMT) plays both a regulatory and protective role in catechol homeostasis. It contributes to the regulation of tissue levels of catecholamines and catecholestrogens (CEs) and, by blocking oxidative metabolism of catechols, prevents endogenous and exogenous catechols from becoming a source of potentially mutagenic electrophiles. Evidence implicating CEs in carcinogenesis, in particular in the hamster kidney model of estrogen-induced cancer, has focused attention on the protective role of COMT in estrogen target tissues. We have previously reported that treating hamsters with estrogens causes translocation of COMT to nuclei of epithelial cells in the renal cortex, the site of CE biosynthesis and where the cancers arise. This finding suggested that nuclear COMT may be a marker of a threat to the genome by catechols, including CEs. It is postulated that CEs play a role in the genesis of breast cancer by contributing to a state of chronic oxidative stress that is presumed to underlie the high incidence of this disease in the United States. Therefore, here we used immunocytochemistry to re-examine human breast parenchyma for nuclear COMT. In addition to confirming previous reports of cytoplasmic COMT in mammary epithelial cells, we identified nuclear COMT in foci of mammary epithelial cells in histologically normal breast tissue of virtually all control (macromastia) and cancer patients and in breast cancer cells. There was no correlation between tissue histology and the numbers of cells with nuclear COMT, the size of foci containing such cells, or intensity of nuclear COMT immunostaining. The focal nature of the phenomenon suggests that nuclear COMT does not serve a housekeeping function but that it reflects a protective response to an increased local catechol load, presumably of CEs and, as such, that it may be a characteristic of the population of women studied who share the same major risk factor for developing breast cancer, that of living in the industrialized West.

No abstract

Glucose utilization and Fos expression were used to compare responses of cerebral structures involved in osmoregulation in virgin and lactating rats given 0.15, 0.85, or 1.5 M NaCl subcutaneously. In virgin animals, glucose utilization increased (P < 0.05) in the supraoptic nuclei (SON), paraventricular nuclei (PVN), and neural lobe (NL) proportionally to the osmotic stimulus (0.15 M NaCl < 0.85 M NaCl < 1.5 M NaCl), whereas metabolism in the median preoptic nucleus (MPO) and median eminence (ME) increased only after 1.5 M NaCl. In lactating rats, enhanced utilization of glucose in response to osmotic stimulation was absent in the PVN (0.85 M NaCl), MPO, and ME or significantly (P < 0.01) reduced (SON, PVN, NL) compared with virgin animals. Glucose utilization in each structure correlated linearly with plasma osmolality but with a lower slope (P < 0.05) in lactating animals. Magnocellular neurons expressing Fos in the SON increased linearly with plasma osmolality and were more numerous (P < 0.05) in control lactating animals but increased less (P < 0.05) than in virgin rats after 0.85 M NaCl. The attenuated magnocellular response during lactation results from reduced afferent activation from osmosensitive forebrain sites.