Rankl Impairs Lactogenic Differentiation Through Inhibition of the Prolactin/Stat5 Pathway at Midgestation.

Prolactin and progesterone both orchestrate the proliferation and differentiation of the mammary gland during gestation. Differentiation of milk secreting alveoli depends on the presence of prolactin receptor, the downstream Jak2-Stat5 pathway and the transcription factor Elf5. A strict regulation of Rank signaling is essential for the differentiation of the mammary gland and in particular for alveolar commitment. Impaired alveologenesis and lactation failure are observed in both, knockout and Rank overexpressing mice; however, the underlying molecular mechanism responsible for these phenotypes remains largely unknown. Using genome-wide expression analyses and functional studies, we show here that Rankl (RL) exposure leads to impaired secretory differentiation of alveolar cells not only in MMTV-RANK but also in wild-type (WT) mammary acini. Conversely, pharmacological blockage of Rank signaling at midgestation in WT mice leads to precocious and exacerbated lactogenesis. Mechanistically, RL negatively regulates Stat5 phosphorylation and Elf5 expression at the onset of lactogenesis. Continuous RL exposure leads to the expansion of basal and bipotent cells in WT and MMTV-RANK acini. Overall, we demonstrate that enhanced Rank signaling impairs secretory differentiation during pregnancy by inhibition of the prolactin/p-Stat5 pathway.

RANK is a poor prognosis marker and a therapeutic target in ER-negative postmenopausal breast cancer.

Despite strong preclinical data, the therapeutic benefit of the RANKL inhibitor, denosumab, in breast cancer patients, beyond the bone, is unclear. Aiming to select patients who may benefit from denosumab, we hereby analyzed RANK and RANKL protein expression in more than 2,000 breast tumors (777 estrogen receptor-negative, ER- ) from four independent cohorts. RANK protein expression was more frequent in ER- tumors, where it associated with poor outcome and poor response to chemotherapy. In ER- breast cancer patient-derived orthoxenografts (PDXs), RANKL inhibition reduced tumor cell proliferation and stemness, regulated tumor immunity and metabolism, and improved response to chemotherapy. Intriguingly, tumor RANK protein expression associated with poor prognosis in postmenopausal breast cancer patients, activation of NFKB signaling, and modulation of immune and metabolic pathways, suggesting that RANK signaling increases after menopause. Our results demonstrate that RANK protein expression is an independent biomarker of poor prognosis in postmenopausal and ER- breast cancer patients and support the therapeutic benefit of RANK pathway inhibitors, such as denosumab, in breast cancer patients with RANK+ ER- tumors after menopause.

An impaired transendothelial migration potential of chronic lymphocytic leukemia (CLL) cells can be linked to ephrin-A4 expression.

Chronic lymphocytic leukemia (CLL) cell migration into lymphoid tissues is an important aspect of the pathobiology of this disease. Here, we investigated the role of ephrin-A4 (EFNA4) in the transendothelial migration (TEM) capacity of CLL and normal B cells through interacting with endothelial EphA2 (erythropoietin-producing hepatocellular carcinoma). CLL cells showed a remarkable impairment in the adhesion to and transmigration through human umbilical vein endothelial cell (HUVEC) monolayers, correlating with their higher EFNA4 expression. In vitro, TEM was mediated by EFNA4 binding to endothelial EphA2 receptor, which is highly expressed in tumor necrosis factor-alpha-activated HUVECs as well as in the CD31(+) endothelial cells of human lymph nodes. The pretreatment of CLL cells with EphA2 homodimers further impaired their adhesion to and transmigration through HUVEC monolayers, whereas pretreatment of HUVECs with EFNA4 homodimers improved those phenomena in both CLL and normal B cells, suggesting that EFNA4 signaling negatively contributed to TEM. In fact, EFNA4 signaling into CLL cells significantly reduced their adhesion to intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and several extracellular matrix molecules and impaired CCL-19-mediated TEM and chemotaxis. Our results suggest that EFNA4-EphA2 interactions are involved in CLL cell trafficking between blood and the tissues and therefore may become a therapeutic target in the future.

Development of Poly (Methyl vinyl ether-alt-maleic acid) Microneedles for Transdermal Delivery of Atorvastatin Calcium.

AIMS: Biodegradable polymeric microneedles containing atorvastatin calcium were developed in order to improve the percutaneous absorption of the drug, useful for the treatment of hypercholesterolemia. BACKGROUND: The use of physical enhancers like microneedles have shown good results to increase the delivery of drugs through the skin, the use of microneedles has very important advantages for transdermal drug delivery, for example, they are painless, easy to use and safe, they increase time interval of drug activity, dose, and reductions in adverse reactions, they also offer, the facility to remove the system instantly of the skin. OBJECTIVE: Develop polymer microneedles loaded with a calcium atorvastatin and evaluate them by Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), bioadhesion, postwetting- bioadhesion, breaking strength, drug release test and in vitro percutaneous absorption studies to demonstrate the use of microneedles atorvastatin is able to cross the skin. METHODS: The microneedles were made with poly (methyl vinyl ether-alt-maleic acid) as biodegradable polymer using the technique of casting in solution in a mold. After solidification these microneedles were characterized by Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM), bioadhesion, post-wetting-bioadhesion, breaking strength, drug release test and in vitro percutaneous absorption studies. RESULTS: In general, the performances were satisfactory for optimal formulation in terms of DSC with no interactions between drug and excipients, SEM shows microneedles with a conical shape, bioadhesion of 1570 g.f, post wetting-bioadhesion of 1503.4 g.f, breaking strength of 1566.7g.f that is sufficient to disrupt Stratum corneum, good drug release and a flux of 33.4 µg/cm2*h with a tLag of 15.14 h for the in vitro percutaneous absorption. CONCLUSION: The results indicate that it is possible to generate microneedles to increase the percutaneous absorption of calcium atorvastatin transdermally, with the potential to be used as an alternative to the oral route for the treatment of dyslipidemias.

Inhibition of RANK signaling in breast cancer induces an anti-tumor immune response orchestrated by CD8+ T cells.

Most breast cancers exhibit low immune infiltration and are unresponsive to immunotherapy. We hypothesized that inhibition of the receptor activator of nuclear factor-κB (RANK) signaling pathway may enhance immune activation. Here we report that loss of RANK signaling in mouse tumor cells increases leukocytes, lymphocytes, and CD8+ T cells, and reduces macrophage and neutrophil infiltration. CD8+ T cells mediate the attenuated tumor phenotype observed upon RANK loss, whereas neutrophils, supported by RANK-expressing tumor cells, induce immunosuppression. RANKL inhibition increases the anti-tumor effect of immunotherapies in breast cancer through a tumor cell mediated effect. Comparably, pre-operative single-agent denosumab in premenopausal early-stage breast cancer patients from the Phase-II D-BEYOND clinical trial (NCT01864798) is well tolerated, inhibits RANK pathway and increases tumor infiltrating lymphocytes and CD8+ T cells. Higher RANK signaling activation in tumors and serum RANKL levels at baseline predict these immune-modulatory effects. No changes in tumor cell proliferation (primary endpoint) or other secondary endpoints are observed. Overall, our preclinical and clinical findings reveal that tumor cells exploit RANK pathway as a mechanism to evade immune surveillance and support the use of RANK pathway inhibitors to prime luminal breast cancer for immunotherapy.

Chromosome 12p Amplification in Triple-Negative/BRCA1-Mutated Breast Cancer Associates with Emergence of Docetaxel Resistance and Carboplatin Sensitivity.

Taxanes are the mainstay of treatment in triple-negative breast cancer (TNBC), with de novo and acquired resistance limiting patient's survival. To investigate the genetic basis of docetaxel resistance in TNBC, exome sequencing was performed on matched TNBC patient-derived xenografts (PDX) sensitive to docetaxel and their counterparts that developed resistance in vivo upon continuous drug exposure. Most mutations, small insertions/deletions, and copy number alterations detected in the initial TNBC human metastatic samples were maintained after serial passages in mice and emergence of resistance. We identified a chromosomal amplification of chr12p in a human BRCA1-mutated metastatic sample and the derived chemoresistant PDX, but not in the matched docetaxel-sensitive PDX tumor. Chr12p amplification was validated in a second pair of docetaxel-sensitive/resistant BRCA1-mutated PDXs and after short-term docetaxel treatment in several TNBC/BRCA1-mutated PDXs and cell lines, as well as during metastatic recurrence in a patient with BRCA1-mutated breast cancer who had progressed on docetaxel treatment. Analysis of clinical data indicates an association between chr12p amplification and patients with TNBC/basal-like breast cancer, a BRCA1 mutational signature, and poor survival after chemotherapy. Detection of chr12p amplification in a cohort of TNBC PDX models was associated with an improved response to carboplatin. Our findings reveal tumor clonal dynamics during chemotherapy treatments and suggest that a preexisting population harboring chr12p amplification is associated with the emergence of docetaxel resistance and carboplatin responsiveness in TNBC/BRCA1-mutated tumors. SIGNIFICANCE: Chr12p copy number gains indicate rapid emergence of resistance to docetaxel and increased sensitivity to carboplatin, therefore sequential docetaxel/carboplatin treatment could improve survival in TNBC/BRCA1 patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/16/4258/F1.large.jpg.

Radiopharmacokinetic and dosimetric parameters of 188Re-lanreotide in athymic mice with induced human cancer tumors.

Radiolabeled peptides, like the somatostatin analogs, have been used for peptide receptor-mediated radionuclide therapy (PRMRT) in metastatic neuroendocrine tumors. The eight amino acid peptide 3-(2-naphthalenyl)-D-alanyl-L-cysteinyl-L-tyrosyl-D-tryptophyl-L-lysyl-L-valyl-L-cysteinyl-L-threoninamide,cyclic(2-->7)-disulfide (9Cl) (lanreotide) was found to bind to the five somatostatin tumor receptors. Lanreotide has been labeled via the bifunctional chelating agent, DOTA, to (111)In, and (90)Y. A direct labeling method was used to label lanreotide with (188)Re. Athymic mice with implanted human cancer tumors (uterine-cervix, renal, and neuroblastoma) were injected with radiochemically pure (188)Re-lanreotide (1.11 MBq). The percent injected activity (%IA/g) from serial blood samples was the input data for the WinNonlin computer program to obtain radiopharmacokinetic parameters. The organs' percent injected activity per gram of tissue (%IA/g) was extrapolated to the weights of a 70 kg male model organs and the number of nuclear transitions (N) were the input for the OLINDA/EXM program to obtain dosimetry estimates. Induced uterine-cervix tumors (HeLa cells) show a mean 2.4 %IA/g uptake up to 24 h and the tumor/blood ratio was over 1.85 (1.5-24 h post-injection) confirming (188)Re-lanreotide remains bound to the tumor. The estimated tumor absorbed dose was 460 mGy/MBq. Human effective dose was 0.0182 mSv/MBq. Therefore, (188)Re-lanreotide is a good candidate for PRMRT and a clinical trial is being planned in order to acquire individual dosimetric data.

EphrinA4 plays a critical role in α4 and αL mediated survival of human CLL cells during extravasation.

A role of endothelial cells in the survival of CLL cells during extravasation is presently unknown. Herein we show that CLL cells but not normal B cells can receive apoptotic signals through physical contact with TNF-α activated endothelium impairing survival in transendothelial migration (TEM) assays. In addition, the CLL cells of patients having lymphadenopathy (LApos) show a survival advantage during TEM that can be linked to increased expression of α4 and αL integrin chains. Within this context, ephrinA4 expressed on the surface of CLL cells sequestrates integrins and inactivates them resulting in reduced adhesion and inhibition of apoptotic/survival signals through them. In agreement, ephrinA4 silencing resulted in increased survival of CLL cells of LApos patients but not LA neg patients. Similarly was observed when a soluble ephrinA4 isoform was added to TEM assays strongly suggesting that accumulation of this isoform in the serum of LApos patients could contribute to CLL cells dissemination and survival in vivo. In supporting, CLL lymphadenopathies showed a preferential accumulation of apoptotic CLL cells around high endothelial venules lacking ephrinA4. Moreover, soluble ephrinA4 isolated from sera of patients increased the number and viability of CLL cells recovered from the lymph nodes of adoptively transferred mice. Finally, we present evidence suggesting that soluble ephrinA4 mediated survival during TEM could enhance a transcellular TEM route of the CLL cells. Together these findings point to an important role of ephrinA4 in the nodal dissemination of CLL cells governing extravasation and survival.

RANK Signaling Blockade Reduces Breast Cancer Recurrence by Inducing Tumor Cell Differentiation.

RANK expression is associated with poor prognosis in breast cancer even though its therapeutic potential remains unknown. RANKL and its receptor RANK are downstream effectors of the progesterone signaling pathway. However, RANK expression is enriched in hormone receptor negative adenocarcinomas, suggesting additional roles for RANK signaling beyond its hormone-dependent function. Here, to explore the role of RANK signaling once tumors have developed, we use the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT), which mimics RANK and RANKL expression patterns seen in human breast adenocarcinomas. Complementary genetic and pharmacologic approaches demonstrate that therapeutic inhibition of RANK signaling drastically reduces the cancer stem cell pool, decreases tumor and metastasis initiation, and enhances sensitivity to chemotherapy. Mechanistically, genome-wide expression analyses show that anti-RANKL therapy promotes lactogenic differentiation of tumor cells. Moreover, RANK signaling in tumor cells negatively regulates the expression of Ap2 transcription factors, and enhances the Wnt agonist Rspo1 and the Sca1-population, enriched in tumor-initiating cells. In addition, we found that expression of TFAP2B and the RANK inhibitor, OPG, in human breast cancer correlate and are associated with relapse-free tumors. These results support the use of RANKL inhibitors to reduce recurrence and metastasis in breast cancer patients based on its ability to induce tumor cell differentiation. Cancer Res; 76(19); 5857-69. ©2016 AACR.

Information and Communication Technologies in the Care of the Elderly: Systematic Review of Applications Aimed at Patients With Dementia and Caregivers.

BACKGROUND: The interest in applying information and communications technology (ICT) in older adult health care is frequently promoted by the increasing and unsustainable costs of health care services. In turn, the unprecedented growth of the elderly population around the globe has urged institutions, companies, industries, and governments to respond to older adults' medical needs. OBJECTIVE: The aim of this review is to systematically identify the opportunities that ICT offers to health services, specifically for patients with dementia and their families. METHODS: A systematic review of the literature about ICT applications that have been developed to assist patients with Alzheimer's disease (AD) and their primary caregivers was conducted. The bibliographic search included works published between January 2005 and July 2015 in the databases Springer Link, Scopus, and Google Scholar. Of the published papers, 902 were obtained in the initial search, of which 214 were potentially relevant. Included studies fulfilled the following inclusion criteria: (1) studies carried out between the years of 2005 and 2015, (2) studies were published in English or Spanish, (3) studies with titles containing the keywords, (4) studies with abstracts containing information on ICT applications and AD, and (5) studies published in indexed journals, proceedings, and book chapters. RESULTS: A total of 26 studies satisfied the inclusion criteria for the current review. Among them, 16 were aimed at the patient with AD and 10 at the primary caregivers and/or family members. The studies targeted applications that included assistive technology (44%, 7/16), telecare (37%, 6/16), and telemedicine (31%, 5/16). The information systems (56%, 9/16) and Internet (44%, 7/16) were the most commonly used enabling technologies for the studies. Finally, areas of attention more covered by the studies were care (56%, 9/16), treatment (56%, 9/16), and management (50%, 8/16). Furthermore, it was found that 20 studies (77%, 8/26) evaluated their ICT applications through carrying out tests with patients with dementia and caregivers. CONCLUSIONS: The key finding of this systematic review revealed that the use of ICT tools can be strongly recommended to be used as a lifestyle in the elderly in order to improve the quality of life for the elderly and their primary caregivers. Since patients with AD are completely dependent in most activities, it is necessary to give attention to their primary caregivers to avoid stress and depression. In addition, the use of ICT in the daily life of caregivers can help them understand the disease process and manage situations in a way that is beneficial for both parties. It is expected that future developments concerning technological projects can support this group of people.

Eph-ephrin bidirectional signaling comes into the context of lymphocyte transendothelial migration.

A better knowledge of the molecular mechanisms that govern leukocyte trafficking is of major relevance for the clinics. Both normal and pathologic extravasation of lymphocytes are a fine-tuned spatio-temporal event of migratory path-finding likely regulated by molecular guidance cues underlying cell movements in other systems. We have recently reported that members of the Eph family of receptor tyrosine kinases, namely EphA2 and one of its ligands, ephrin-A4 (EFNA4) can mediate in the traffic of chronic lymphocytic leukemia (CLL) cells and presumably of normal B cells between the blood and the tissues. The importance of EphA2-EFNA4 interactions at the endothelium-lymphocyte interface during TEM could rely on their attractive/repulsive properties. In the present work, we expand on those results by including additional insights and new suggestions for future studies that discuss the relevance of these molecules in overall cell adhesion dynamic events.

Therapeutic 188Re-lanreotide: determination of radiopharmacokinetic parameters in rats.

OBJECTIVES: The radiopharmacokinetic parameters of the therapeutic radiopharmaceutical (188)Re-lanreotide were compared in rats implanted with hepatocarcinoma tumours (n= 18) and healthy rats (n= 18). METHODS: Rats were injected with approximately 1.8 MBq (188)Re-lanreotide (0.1 ml) via the tail vein and blood samples were obtained. The activity per gram of tissue (%IA/g) was calculated and the radiopharmacokinetic parameters determined. Data were fitted using a two-compartment model. KEY FINDINGS: Significant differences were found between healthy and hepatoma rats for beta elimination half-life (22.56 vs 48.14 h); transference constants K(10) (k(e)) (6.44 vs 3.05 h(-1)) and K(12) (2.76 vs 7.09 h(-1)); volume of distribution (2.06 vs 5.45 ml); mean residence time (66.58 vs 95.50 h) and apparent volume of distribution at steady state (131.30 vs 810.37 ml). The tumour/organ ratios after 24 h were 11.20 for tumour/muscle, 8.00 for tumour/liver and 7.72 for tumour/bone. The scintigraphic images obtained therefore had high resolution. CONCLUSIONS: (188)Re-lanreotide had a prolonged beta elimination half-life and increased volume of distribution in rats with hepatocellular carcinoma. This may be beneficial in the diagnosis and therapy of metastatic lesions in patients with cancer.

Expression profile of Eph receptors and ephrin ligands in healthy human B lymphocytes and chronic lymphocytic leukemia B-cells.

Increasing information relates some Eph receptors and their ligands, ephrins (EFN), with the immune system. Herein, we found that normal B-cells from peripheral blood (PB) and lymph nodes (LN) showed a differential expression of certain Eph/EFN members, some of them being modulated upon in vitro stimulation including EFNA1, EFNA4, EphB6 and EphA10. In contrast, PB CLL B-cells showed a more heterogeneous Eph/EFN profile than their normal PB B-cell counterparts, expressing Eph/EFN members frequently found within the LN and activated B-cells, specially EFNA4, EphB6 and EphA10. Two of them, EphB6 and EFNA4 were further related with the clinical course of CLL patients. EphB6 expression correlated with a high content of ZAP-70 mRNA and a poor prognosis. High serum levels of a soluble EFNA4 isoform positively correlated with increasing peripheral blood lymphocyte counts and lymphadenopathy. These findings suggest that Eph/EFN might be relevant in normal B-cell biology and could represent new potential prognostic markers and therapeutic targets for CLL.