NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

Impact of Anti-Angiogenic Treatment on Bone Vascularization in a Murine Model of Breast Cancer Bone Metastasis Using Synchrotron Radiation Micro-CT.

Bone metastases are frequent complications of breast cancer, facilitating the development of anarchic vascularization and induce bone destruction. Therefore, anti-angiogenic drugs (AAD) have been tested as a therapeutic strategy for the treatment of breast cancer bone metastasis. However, the kinetics of skeletal vascularization in response to tumor invasion under AAD is still partially understood. Therefore, the aim of this study was to explore the effect of AAD on experimental bone metastasis by analyzing the three-dimensional (3D) bone vasculature during metastatic formation and progression. Seventy-three eight-week-old female mice were treated with AAD (bevacizumab, vatalanib, or a combination of both drugs) or the vehicle (placebo) one day after injection with breast cancer cells. Mice were sacrificed eight or 22 days after tumor cell inoculation (time points T1 and T2, respectively). Synchrotron radiation microcomputed tomography (SR-µCT) was used to image bone and blood vessels with a contrast agent. Hence, 3D-bone and vascular networks were simultaneously visualized and quantitatively analyzed. At T1, the trabecular bone volume fraction was significantly increased (p < 0.05) in the combined AAD-treatment group, compared to the placebo- and single AAD-treatment groups. At T2, only the bone vasculature was reduced in the combined AAD-treatment group (p < 0.05), as judged by measurement of the blood vessel thickness. Our data suggest that, at the early stage, combined AAD treatment dampens tumor-induced bone resorption with no detectable effects on bone vessel organization while, at a later stage, it affects the structure of bone microvascularization.

Integrin alpha5 in human breast cancer is a mediator of bone metastasis and a therapeutic target for the treatment of osteolytic lesions.

Bone metastasis remains a major cause of mortality and morbidity in breast cancer. Therefore, there is an urgent need to better select high-risk patients in order to adapt patient's treatment and prevent bone recurrence. Here, we found that integrin alpha5 (ITGA5) was highly expressed in bone metastases, compared to lung, liver, or brain metastases. High ITGA5 expression in primary tumors correlated with the presence of disseminated tumor cells in bone marrow aspirates from early stage breast cancer patients (n = 268; p = 0.039). ITGA5 was also predictive of poor bone metastasis-free survival in two separate clinical data sets (n = 855, HR = 1.36, p = 0.018 and n = 427, HR = 1.62, p = 0.024). This prognostic value remained significant in multivariate analysis (p = 0.028). Experimentally, ITGA5 silencing impaired tumor cell adhesion to fibronectin, migration, and survival. ITGA5 silencing also reduced tumor cell colonization of the bone marrow and formation of osteolytic lesions in vivo. Conversely, ITGA5 overexpression promoted bone metastasis. Pharmacological inhibition of ITGA5 with humanized monoclonal antibody M200 (volociximab) recapitulated inhibitory effects of ITGA5 silencing on tumor cell functions in vitro and tumor cell colonization of the bone marrow in vivo. M200 also markedly reduced tumor outgrowth in experimental models of bone metastasis or tumorigenesis, and blunted cancer-associated bone destruction. ITGA5 was not only expressed by tumor cells but also osteoclasts. In this respect, M200 decreased human osteoclast-mediated bone resorption in vitro. Overall, this study identifies ITGA5 as a mediator of breast-to-bone metastasis and raises the possibility that volociximab/M200 could be repurposed for the treatment of ITGA5-positive breast cancer patients with bone metastases.

ERRα Expression in Bone Metastases Leads to an Exacerbated Antitumor Immune Response.

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFß3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFß signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.

Efecto de los factores relacionados al cuidado de la diabetes mellitus tipo 2 y motivos de no adhesión en Argentina / Effect of factors related to the care of type 2 diabetes mellitus and reasons for non-adherence in Argentina

Introducción: analizar la injerencia de la adherencia al tratamiento en la evaluación clínica optimiza el uso de pruebas y evita intensificar tratamientos que pueden aumentar riesgos en los pacientes. Conocer sobre las variables influyentes implica el conocimiento y el control de factores relativos al cuidado de la diabetes mellitus tipo 2 (DM2). Objetivos: analizar el efecto de la adherencia al tratamiento en adultos con DM2 en Argentina. Materiales y métodos: estudio transversal. Se incluyeron 1.520 pacientes adultos con DM2 con tres o más meses de antigüedad de enfermedad residentes en Argentina. Se aplicó un cuestionario validado autorreferido. Se evaluó el grado de adherencia reportada, las recomendaciones dadas por el equipo de salud, las formas de adhesión y los motivos de incumplimiento por medio del Summary of Diabetes Self-Care Activities (SDSCA). Resultados: representando diferentes regiones geográficas, la media de adherencia general para todas las características fue 4,32 (61,71%); los peores puntajes para la adherencia correspondieron a la actividad física, el monitoreo glucémico y la dieta en orden ascendente. Se observaron carencias en las recomendaciones por parte del equipo de salud al momento de indicar medidas higiénico dietéticas. Conclusiones: los resultados nacionales observados guardan similitud en la dieta, la actividad física y la disminución en el consumo de tabaco con respecto a los datos prevalentes proveniente de la Cuarta Encuesta Nacional de Factores de Riesgo

Introduction: analyzing the interference of adherence to treatment in clinical evaluation optimizes the use of tests and avoids intensifying treatments that may increase risks in patients. Knowing about the influential variables implies the knowledge and control of factors related to the care of diabetes mellitus type 2 (T2D). Objectives: to analyze the effect of adherence to treatment in adults with T2D in Argentina. Materials and methods: transversal study. We included 1.520 adult patients with T2D with 3 or more months of illness in Argentina. A validated self-reported questionnaire was applied. The degree of adherence reported, the recommendations given by the health team, the forms of adherence and the reasons for non-compliance through the Summary of Diabetes Self-Care Activities (SDSCA) were evaluated. Results: representing different geographical regions, the mean of general adherence for all the characteristics was 4.32 (61.71%); the worst scores for adherence corresponded to physical activity, glycemic monitoring and diet in ascending order. There were shortcomings in the recommendations by the health team when indicating hygienic dietary measures. Conclusions: the observed national results are similar in diet, physical activity and the decrease in tobacco consumption with respect to prevailing data from the Fourth National Survey of Risk Factors.

Calidad de vida y prestaciones en salud de pacientes con diabetes mellitus tipo 2 según región geográfica en Argentina / Quality of life and health benefits in patients with type 2 diabetes according to geography region in Argentina

Introducción: la adherencia al tratamiento de la diabetes mellitus tipo 2 (DM2) en el adulto es un tema complejo y multifactorial que aúna aspectos propios de la enfermedad como no propios, los cuales impactan en la calidad de vida. En Argentina se presentan grandes dificultades al costear la totalidad del tratamiento de la enfermedad. El impacto del ingreso sobre el gasto en salud en DM2 es mayor para los niveles más bajos de ingresos. Objetivos: analizar en adultos con DM2 en Argentina diferencias en variables de calidad de vida y objetivos metabólicos según regiones geográficas y prestación en salud; conocer el gasto de bolsillo en salud y los costos en salud vinculados a DM2; establecer la asociación entre el gasto de bolsillo con variables socioeconómicas y el cumplimiento de los objetivos metabólicos. Materiales y métodos: sobre ocho regiones geográficas de la República Argentina se incluyeron 1.520 pacientes adultos con DM2 y se los clasificó según prestación en salud (sin prestación, con prestación o jubilados). Se aplicó cuestionario validado autorreferido. Resultados: se encontraron diferencias significativas por región geográfica para jubilación prematura por DM2, pérdida de percepción de calidad de vida y cumplimiento de los objetivos metabólicos. Se encontró significancia estadística del gasto de bolsillo con la presencia de subsidio familiar, la jubilación prematura por DM2 y el cumplimiento de los objetivos metabólicos. Conclusiones: se observó disparidad entre las variables de calidad de vida, el gasto en salud y la percepción de subsidios

Introduction: adherence to treatment of diabetes mellitus type 2 (DT2) in the adult is a complex and multifactorial issue that combines aspects of the disease as not own, which impact on the quality of life. In Argentina there are great difficulties in paying for the entire treatment of the disease. The impact of income on health spending in DT2 is greater for lower income levels. Objectives: to analyze differences in quality of life and metabolic objectives according to geographic regions and health benefits in adults with DT2 in Argentina; know the out of pocket expenses in health and health costs linked to DT2; establish an association between out of pocket spending with socioeconomic variables and compliance with metabolic objectives. Materials and methods: over eight geographic regions of the Argentine Republic, 1.520 adult patients with DT2 were classified according to health benefit (without benefit, with benefits or retirees). Validated self-reported questionnaire was applied. Results: significant differences were found by geographic region for premature retirement due to DT2, the loss of perception of quality of life and the fulfillment of metabolic objectives. Statistical significance of out-of-pocket spending was found with the presence of family allowance, early retirement by DT2 and compliance with metabolic objectives. Conclusions: disparity between the variables of quality of life, health expenditure and perception of subsidies was observed

Adherencia al tratamiento en pacientes con diabetes mellitus tipo 2 en Argentina durante 2015 / Adherence to treatment in patients with type 2 diabetes melltius in Argentina during 2015

Introducción: la interrupción en la adherencia a los tratamientos prescriptos genera obstáculos importantes los cuales impactan negativamente en los indicadores de salud. Objetivos: evaluar la adherencia al tratamiento en pacientes adultos con diabetes mellitus tipo 2 (DM2) en Argentina, y establecer la asociación con el tipo de prestación de atención en salud y la ubicación geográfica. Materiales y métodos: se incluyeron 1.520 individuos pertenecientes a ocho regiones diferentes de la República Argentina durante 2015. Se realizó un estudio transversal. Se aplicó un cuestionario validado autorreferido de opciones múltiples con respuestas policotómicas de puntuación simple. Se empleó una escala cuantitativa y un análisis multivariado de componentes principales. Resultados: la edad media fue de 60,2 años y la antigüedad de DM2 referida correspondió a 10,1 años. La media de HbA1c fue de 7,85%. La media de adherencia general para todas las características fue 4,32 (61,71%). Los resultados de adherencia encontrados se asemejan a otros países en desarrollo a excepción del cuidado de los pies. El factor cuidado de los pies en nuestro país reporta escalas de mayor adherencia. Se detectaron resultados tal vez redundantes como la mayor adherencia al monitoreo en las terapias de uso con insulina, y menores para dieta, ejercicio y solo uso de antidiabéticos orales (ADO), así como también la mayor adherencia en medicación en las terapias con insulina e insulina más ADO. Conclusiones: son alarmantes las bajas escalas referidas al grupo con solo dieta y ejercicio

Introduction: the interruption in the adherence to the prescribed treatments generates important obstacles which negatively impact on health indicators. Objectives: to evaluate adherence to treatment in adult patients with diabetes mellitus type 2 (DT2) in Argentina; associate with the type of health care provision and location. Materials and methods: we included 1.520 individuals belonging to eight different geographical regions of the Argentine Republic during 2015. A cross-sectional study was conducted. A self-reported validated questionnaire of multiple options with simple scoring polycotomic responses was applied. A quantitative scale and a multivariate analysis of main components were applied. Results: the average age was 60.2 years and the seniority of the referred DT2 corresponded to 10.1 years. The average HbA1c was 7.85%. The average general adherence for all characteristics was 4.32 (61.71%). The adherence results found are similar to other developing countries except for the care of the feet. The care factor of the feet in our country report scales of greater adherence. Redundant results are observed, such as greater adherence to monitoring in insulin therapy and less for diet and exercise and only oral antidiabetic drugs (ADO), as well as greater medication adherence in insulin and insulin therapies more ADO. Conclusions: the low scales referred to the group with only diet and exercise are alarming

The C-Terminal Intact Forms of Periostin (iPTN) Are Surrogate Markers for Osteolytic Lesions in Experimental Breast Cancer Bone Metastasis.

Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (- 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer.

Overexpression of a functional calcium-sensing receptor dramatically increases osteolytic potential of MDA-MB-231 cells in a mouse model of bone metastasis through epiregulin-mediated osteoprotegerin downregulation.

INTRODUCTION AND AIMS: Osteolytic bone metastases are observed in advanced cases of breast cancer. In vitro data suggest that the activity of the calcium-sensing receptor (CaSR) expressed by metastatic cells could potentiate their osteolytic potential. This study aimed to demonstrate in vivo the involvement of the CaSR in breast cancer cells osteolytic potential and to identify potential targets linked to CaSR activity. METHODS AND RESULTS: MDA-MB-231 stably transfected with plasmids containing either a full-length wild-type CaSR (CaSR-WT), or a functionally inactive dominant negative mutant (CaSR-DN) or an empty vector (EV) were intratibially injected into Balb/c-Nude mice. X-ray analysis performed 19 days after injection showed a dramatic increase of osteolytic lesions in mice injected with CaSR-WT-transfected cells as compared to mice injected with EV- or CaSR-DN-transfected cells. This was associated with decreased BV/TV ratio and increased tumor burden. Epiregulin, an EGF-like ligand, was identified by a DNA microarray as a possible candidate involved in CaSR-mediated osteolysis. Indeed, in vitro, CaSR overexpression increased both epiregulin expression and secretion as compared to EV- or CaSR-DN-transfected cells. Increased epiregulin expression was also detected in osteolytic bone lesions from mice injected with CaSR-WT-transfected MDA-MB-231. In vitro, exposure of osteoblastic cells (HOB and SaOS2) to exogenous epiregulin significantly decreased OPG mRNA expression. Exposure of osteoblastic cells to conditioned media prepared from CaSR-WT-transfected cells also decreased OPG expression. This effect was partially blocked after addition of an anti-epiregulin antibody. CONCLUSIONS: Overexpression of a functional CaSR in metastatic breast cancer cells dramatically amplifies their osteolytic potential through epiregulin-mediated OPG downregulation.

Unseeded Inertial Cavitation for Enhancing the Delivery of Chemotherapies: A Safety Study.

Acoustic cavitation can improve local drug delivery in tumors. Without injected external nucleation agents, initiating inertial cavitation requires high negative pressures, which can lead to biological damage. In the present study, unseeded inertial cavitation was obtained in vivo using confocal beams, and the effect of these exposure conditions was assessed on drug structure and activity, shallow tissues and growth of breast tumors. No change was observed in the structure and cytotoxicity of doxorubicin. Experiments were conducted on healthy rats, exposing the thigh and abdomen. Histologic analyses at 72 h and 2 weeks post-treatment demonstrated a modest impact on tissues. Syngeneic 4 T1 breast tumors in mice were sonicated. Immunohistochemical analyses showed that ultrasound did not impact vascular density, proliferation and apoptosis of cancer cells. In addition, ultrasound did not negatively modify cancer cell spreading to the lungs and bone marrow. This provides evidence that these particular parameters can be used safely in vivo.

Inhibition of DNA-repair genes Ercc1 and Mgmt enhances temozolomide efficacy in gliomas treatment: a pre-clinical study.

Gliomas are the most common primary brain tumors. To date, therapies do not allow curing patients, and glioblastomas (GBMs) are associated with remarkably poor prognosis. This situation is at least partly due to intrinsic or acquired resistance to treatment, especially to chemotherapy. In 2005, temozolomide (TMZ) has become the first chemotherapeutic drug validated for GBM. Nevertheless TMZ efficacy depends on Mgmt status. While the methylation of Mgmt promoter was considered so far as a prognostic marker, its targeting is becoming an effective therapeutic opportunity. Thus, arrival of both TMZ and Mgmt illustrated that considerable progress can still be realized by optimizing adjuvant chemotherapy. A part of this progress could be accomplished in the future by overcoming residual resistance. The aim of the present study was to investigate the involvement of a set of other DNA-repair genes in glioma resistance to temozolomide. We focused on DNA-repair genes located in the commonly deleted chromosomal region in oligodendroglioma (1p/19q) highly correlated with patient response to chemotherapy. We measured effects of inhibition of ten DNA-repair genes expression using siRNAs on astrocytoma cell response to cisplatin (CDDP) and TMZ. SiRNAs targeting ercc1, ercc2, mutyh, and pnkp significantly sensitized cells to chemotherapy, increasing cell death by up to 25%. In vivo we observed a decrease of subcutaneous glioma tumor growth after injection of siRNA in conjunction with absorption of TMZ. We demonstrated in this pre-clinical study that targeting of DNA-repair genes such as Ercc1 could be used as an adjuvant chemosensitization treatment, similarly to Mgmt inhibition.