Carvedilol blocks neural regulation of breast cancer progression in vivo and is associated with reduced breast cancer mortality in patients.

PURPOSE: The sympathetic nervous system drives breast cancer progression through ß-adrenergic receptor signalling. This discovery has led to the consideration of cardiac ß-blocker drugs as novel strategies for anticancer therapies. Carvedilol is a ß-blocker used in the management of cardiovascular disorders, anxiety, migraine and chemotherapy-induced cardiotoxicity. However, little is known about how carvedilol affects cancer-related outcomes. METHODS: To address this, we investigated the effects of carvedilol on breast cancer cell lines, in mouse models of breast cancer and in a large cohort of patients with breast cancer (n = 4014). RESULTS: Treatment with carvedilol blocked the effects of sympathetic nervous system activation, reducing primary tumour growth and metastasis in a mouse model of breast cancer and preventing invasion by breast cancer cell lines. A retrospective analysis found that women using carvedilol at breast cancer diagnosis (n = 136) had reduced breast cancer-specific mortality compared with women who did not (n = 3878) (5-year cumulative incidence of breast cancer deaths: 3.1% versus 5.7%; p = 0.024 and 0.076 from univariate and multivariable analyses, respectively) after a median follow-up of 5.5 years. CONCLUSIONS: These findings provide a rationale to further explore the use of the ß-blocker carvedilol as a novel strategy to slow cancer progression.

Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations.

G protein-coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous ß2-adrenergic receptor (ß2AR) and muscarinic acetylcholine M3 receptor (M3R) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal-regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations.

ß2-Adrenoceptors on tumor cells play a critical role in stress-enhanced metastasis in a mouse model of breast cancer.

Chronic stress accelerates metastasis - the main cause of death in cancer patients - through the activation of ß-adrenoceptors (ßARs). We have previously shown that ß2AR signaling in MDA-MB-231(HM) breast cancer cells, facilitates invadopodia formation and invasion in vitro. However, in the tumor microenvironment where many stromal cells also express ßAR, the role of ß2AR signaling in tumor cells in metastasis is unclear. Therefore, to investigate the contribution of ß2AR signaling in tumor cells to metastasis in vivo, we used RNA interference to generate MDA-MB-231(HM) breast cancer cells that are deficient in ß2AR. ß2AR knockdown in tumor cells reduced the proportion of cells with a mesenchymal-like morphology and, as expected, reduced tumor cell invasion in vitro. Conversely, overexpression of ß2AR in low metastatic MCF-7 breast cancer cells induced an invasive phenotype. Importantly, we found that knockdown of ß2AR in tumor cells significantly reduced the impact of stress on metastasis in vivo. These findings highlight a crucial role for ß2AR tumor cell signaling in the adverse effects of stress on metastasis, and indicate that it may be necessary to block ß2AR on tumor cells to fully control metastatic progression.

The ß2-adrenoceptor activates a positive cAMP-calcium feedforward loop to drive breast cancer cell invasion.

Activation of the sympathetic nervous system by stress increases breast cancer metastasis in vivo. Preclinical studies suggest that stress activates ß-adrenoceptors (ßARs) to enhance metastasis from primary tumors and that ß-blockers may be protective in breast cancer. However, the subtype of ßAR that mediates this effect, as well as the signaling mechanisms underlying increased tumor cell dissemination, remain unclear. We show that the ß2AR is the only functionally relevant ßAR subtype in the highly metastatic human breast cancer cell line MDA-MB-231HM. ß2AR activation results in elevated cAMP (formoterol pEC50 9.86 ± 0.32), increased intracellular Ca(2+) (formoterol pEC50 8.20 ± 0.33) and reduced phosphorylated ERK (pERK; formoterol pIC50 11.62 ± 0.31). We demonstrate that a highly amplified positive feedforward loop between the cAMP and Ca(2+) pathways is responsible for efficient inhibition of basal pERK. Importantly, activation of the ß2AR increased invasion (formoterol area under the curve [AUC] relative to vehicle: 1.82 ± 0.36), which was dependent on the cAMP/Ca(2+) loop (formoterol AUC in the presence of 2'5'-dideoxyadenosine 0.64 ± 0.03, or BAPTA-AM 0.45 ± 0.23) but independent of inhibition of basal pERK1/2 (vehicle AUC with U0126 0.60 ± 0.30). Specifically targeting the positive feedforward cAMP/Ca(2+) loop may be beneficial for the development of therapeutics to slow disease progression in patients with breast cancer.

ß2-adrenoceptor signaling regulates invadopodia formation to enhance tumor cell invasion.

INTRODUCTION: For efficient metastatic dissemination, tumor cells form invadopodia to degrade and move through three-dimensional extracellular matrix. However, little is known about the conditions that favor invadopodia formation. Here, we investigated the effect of ß-adrenoceptor signaling - which allows cells to respond to stress neurotransmitters - on the formation of invadopodia and examined the effect on tumor cell invasion. METHODS: To characterize the molecular and cellular mechanisms of ß-adrenergic signaling on the invasive properties of breast cancer cells, we used functional cellular assays to quantify invadopodia formation and to evaluate cell invasion in two-dimensional and three-dimensional environments. The functional significance of ß-adrenergic regulation of invadopodia was investigated in an orthotopic mouse model of spontaneous breast cancer metastasis. RESULTS: ß-adrenoceptor activation increased the frequency of invadopodia-positive tumor cells and the number of invadopodia per cell. The effects were selectively mediated by the ß2-adrenoceptor subtype, which signaled through the canonical Src pathway to regulate invadopodia formation. Increased invadopodia occurred at the expense of focal adhesion formation, resulting in a switch to increased tumor cell invasion through three-dimensional extracellular matrix. ß2-adrenoceptor signaling increased invasion of tumor cells from explanted primary tumors through surrounding extracellular matrix, suggesting a possible mechanism for the observed increased spontaneous tumor cell dissemination in vivo. Selective antagonism of ß2-adrenoceptors blocked invadopodia formation, suggesting a pharmacological strategy to prevent tumor cell dissemination. CONCLUSION: These findings provide insight into conditions that control tumor cell invasion by identifying signaling through ß2-adrenoceptors as a regulator of invadopodia formation. These findings suggest novel pharmacological strategies for intervention, by using ß-blockers to target ß2-adrenoceptors to limit tumor cell dissemination and metastasis.

Involvement of insulin-like growth factor binding protein-3 in peroxisome proliferator-activated receptor gamma-mediated inhibition of breast cancer cell growth.

We have previously reported that insulin-like growth factor binding protein-3 (IGFBP-3), a protein with dichotomous effects on both cell proliferation and cell survival, interacts with peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits adipogenic PPARγ signaling. We now show that IGFBP-3 and PPARγ interact in breast cancer cells, through amino- and carboxyl-terminal residues of IGFBP-3. IGFBP-3 and the PPARγ ligands, rosiglitazone or 15-deoxy-Δ(12,14)-prostaglandin J2, separately inhibited the proliferation of MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cells. However, growth inhibition by IGFBP-3 and PPARγ ligand combined was greater than by either alone. Two IGFBP-3 mutants with reduced PPARγ binding caused no growth inhibition when used alone and abolished the inhibitory effect of rosiglitazone when used in combination with PPARγ ligand. Cell growth inhibition by PPARγ ligands was substantially blocked by IGFBP-3 siRNA and restored by exogenous IGFBP-3. We conclude that the interaction between IGFBP-3 and PPARγ is important for the growth-inhibitory effect of PPARγ ligands in human breast cancer cells, suggesting that IGFBP-3 expression by breast tumors may regulate their sensitivity toward PPARγ ligands.

Mutations in KCNJ5 determines presentation and likelihood of cure in primary hyperaldosteronism.

INTRODUCTION: Primary hyperaldosteronism (PA) is a common cause of secondary hypertension. Two recurrent mutations (G151R and L168R) in the potassium channel gene KCNJ5 have been identified that affect the Kir3.4 potassium channel found in the cells of the zona glomerulosa of the adrenal gland. The aim of this study was to determine the prevalence of KCNJ5 mutations in an Australian cohort of patients and to correlate these findings with clinical outcome data, in order to describe the clinical impact on patients who harbour this mutation. METHODS: Direct Sanger sequencing for KCNJ5 on DNA from adrenal tumour tissue of 83 patients with PA in a cohort study was undertaken and mutation status correlated with clinical outcome data. RESULTS: Seventy-one of 83 patients (86%) had adrenocortical adenomas and 12 patients (14%) had bilateral adrenal hyperplasia. A total of 34 (41%) patients were found to have heterozygous somatic mutations in KCNJ5, G151R and L168R. No germ line mutations were identified. Patients with mutations were predominately female (68% versus 49%) and significantly younger at presentation (48 versus 55 years). When correlated with clinical data, our results demonstrated that patients with KCNJ5 mutations were more likely to be cured following surgery without the requirement for ongoing medications. CONCLUSIONS: Our findings in a large Australian cohort show that patients with mutations in KCNJ5 present earlier with the signs and symptoms of PA benefit from surgical intervention. Moreover, our results highlight the importance of a thorough workup and management plan for younger patients who present with hypertension.

Breast cancer-associated fibroblasts induce epithelial-to-mesenchymal transition in breast cancer cells.

Cancer-associated fibroblasts (CAFs) play a role in tumour initiation and progression, possibly by inducing epithelial-to-mesenchymal transition (EMT), a series of cellular changes that is known to underlie the process of metastasis. The aim of this study was to determine whether CAFs and surrounding normal breast fibroblasts (NBFs) are able to induce EMT markers and functional changes in breast epithelial cancer cells. Matched pairs of CAFs and NBFs were established from fresh human breast cancer specimens and characterised by assessment of CXCL12 levels, α-smooth muscle actin (α-SMA) levels and response to doxorubicin. The fibroblasts were then co-cultured with MCF7 cells. Vimentin and E-cadherin expressions were determined in co-cultured MCF7 cells by immunofluorescence and confocal microscopy as well as by western blotting and quantitative PCR. Co-cultured MCF7 cells were also assessed functionally by invasion assay. CAFs secreted higher levels of CXCL12 and expressed higher levels of α-SMA compared with NBFs. CAFs were also less sensitive to doxorubicin as evidenced by less H2AX phosphorylation and reduced apoptosis on flow cytometric analysis of Annexin V compared with NBFs. When co-cultured with MCF7 cells, there was greater vimentin and less E-cadherin expression as well as greater invasiveness in MCF7 cells co-cultured with CAFs compared with those co-cultured with NBFs. CAFs have the ability to induce a greater degree of EMT in MCF7 cell lines, indicating that CAFs contribute to a more malignant breast cancer phenotype and their role in influencing therapy resistance should therefore be considered when treating breast cancer.

Genetic requirements for replication initiation of the staphylococcal multiresistance plasmid pSK41.

Replication of staphylococcal multiresistance plasmid pSK41 is initiated by binding of the replication initiator protein (Rep) to the Rep boxes, a series of four direct repeats located centrally within the rep gene. A Staphylococcus aureus strain was engineered to provide Rep in trans, allowing localization of the pSK41 origin of replication (oriV) to a 185 bp segment, which included the Rep boxes and a series of downstream direct repeats. Deletion analysis of individual Rep boxes revealed that all four Rep boxes are required for maximum origin activity, with the deletion of one or more Rep boxes having a significant effect on the proficiency of replication. However, a hierarchy of importance was identified among the Rep boxes, which appears to be mediated by the minor sequence variations that exist between them. DNA binding studies with truncated Rep proteins have enabled the DNA binding domain to be localized to the N-terminal 134 amino acids of the protein.