Abrogating phosphorylation of eIF4B is required for EGFR and mTOR inhibitor synergy in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) patients suffer from a highly malignant and aggressive disease. They have a high rate of relapse and often develop resistance to standard chemotherapy. Many TNBCs have elevated epidermal growth factor receptor (EGFR) but are resistant to EGFR inhibitors as monotherapy. In this study, we sought to find a combination therapy that could sensitize TNBC to EGFR inhibitors. Phospho-mass spectrometry was performed on the TNBC cell line, BT20, treated with 0.5 µM gefitinib. Immunoblotting measured protein levels and phosphorylation. Colony formation and growth assays analyzed the treatment on cell proliferation, while MTT assays determined the synergistic effect of inhibitor combination. A Dual-Luciferase reporter gene plasmid measured translation. All statistical analysis was done on CalucuSyn and GraphPad Prism using ANOVAs. Phospho-proteomics identified the mTOR pathway to be of interest in EGFR inhibitor resistance. In our studies, combining gefitinib and temsirolimus decreased cell growth and survival in a synergistic manner. Our data identified eIF4B, as a potentially key fragile point in EGFR and mTOR inhibitor synergy. Decreased eIF4B phosphorylation correlated with drops in growth, viability, clonogenic survival, and cap-dependent translation. Taken together, these data suggest EGFR and mTOR inhibitors abrogate growth, viability, and survival via disruption of eIF4B phosphorylation leading to decreased translation in TNBC cell lines. Further, including an mTOR inhibitor along with an EGFR inhibitor in TNBC with increased EGFR expression should be further explored. Additionally, translational regulation may play an important role in regulating EGFR and mTOR inhibitor synergy and warrant further investigation.

Holistic Physical Assessment: A New Framework for Nursing Practice Honoring the Whole Person.

Scholars have argued that standard physical assessment is based on a medical framework instead of nursing theories and philosophy. We argue that standard physical assessment is task-oriented, lacking emphasis on the whole-person and neglecting a least-to-most invasive assessment process. While holistic health assessment is person-centered and multidimensional; addressing aspects such as physical, emotional, and spiritual well-being, there is not a comprehensive explanation on how to holistically perform the physical examination portion of a holistic health assessment. Proposed is a new framework for holistic physical assessment in the hospital environment, honoring the whole-person, their background, and lived experiences. The framework builds on the holistic nursing philosophy, integrative nursing principles, and trauma-informed approach, and systematically arranges physical assessment from least invasive to most. The seven sequential assessment steps incorporate self-care and awareness into the preparation and closure of the assessment-(1) the self, (2) the space, (3) least invasive, (4) moderate invasive, (5) most invasive, (6) the space, and (7) the self. This framework can have significant implications for nursing practice and education settings, providing valuable guidance for a holistic physical assessment that emphasizes whole-person care.

Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met.

INTRODUCTION: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown clinical efficacy in lung, colon, and pancreatic cancers. In lung cancer, resistance to EGFR TKIs correlates with amplification of the hepatocyte growth factor (HGF) receptor tyrosine kinase Met. Breast cancers do not respond to EGFR TKIs, even though EGFR is overexpressed. This intrinsic resistance to EGFR TKIs in breast cancer does not correlate with Met amplification. In several tissue monoculture models of human breast cancer, Met, although expressed, is not phosphorylated, suggesting a requirement for a paracrine-produced ligand. In fact, HGF, the ligand for Met, is not expressed in epithelial cells but is secreted by fibroblasts in the tumor stroma. We have identified a number of breast cancer cell lines that are sensitive to EGFR TKIs. This sensitivity is in conflict with the observed clinical resistance to EGFR TKIs in breast cancers. Here we demonstrate that fibroblast secretion of HGF activates Met and leads to EGFR/Met crosstalk and resistance to EGFR TKIs in triple-negative breast cancer (TNBC). METHODS: The SUM102 and SUM149 TNBC cell lines were used in this study. Recombinant HGF as well as conditioned media from fibroblasts expressing HGF were used as sources for Met activation. Furthermore, we co-cultured HGF-secreting fibroblasts with Met-expressing cancer cells to mimic the paracrine HGF/Met pathway, which is active in the tumor microenvironment. Cell growth, survival, and transformation were measured by cell counting, clonogenic and MTS assays, and soft agar colony formation, respectively. Student's t test was used for all statistical analysis. RESULTS: Here we demonstrate that treatment of breast cancer cells sensitive to EGFR TKIs with recombinant HGF confers a resistance to EGFR TKIs. Interestingly, knocking down EGFR abrogated HGF-mediated cell survival, suggesting a crosstalk between EGFR and Met. HGF is secreted as a single-chain pro-form, which has to be proteolytically cleaved in order to activate Met. To determine whether the proteases required to activate pro-HGF were present in the breast cancer cells, we utilized a fibroblast cell line expressing pro-HGF (RMF-HGF). Addition of pro-HGF-secreting conditioned fibroblast media to TNBC cells as well as co-culturing of TNBC cells with RMF-HGF fibroblasts resulted in robust phosphorylation of Met and stimulated proliferation in the presence of an EGFR TKI. CONCLUSIONS: Taken together, these data suggest a role for Met in clinical resistance to EGFR TKIs in breast cancer through EGFR/Met crosstalk mediated by tumor-stromal interactions.

Reduced-dose rituximab in rheumatoid arthritis: efficacy depends on degree of B cell depletion.

OBJECTIVE: Studies comparing 500 mg rituximab and 1,000 mg rituximab doses in rheumatoid arthritis have yielded conflicting data on clinical outcomes, but in all of these studies a subgroup of patients has had excellent responses at the lower dose. Historically, it was considered that rituximab uniformly depleted B cells at both doses. Using highly sensitive assays, we have shown that B cell depletion is variable and predictive of clinical response. Using the same techniques, we undertook the present study to test the hypothesis that the level of B cell depletion, rather than the rituximab dose, determines clinical response. METHODS: Nineteen patients were treated with two 500-mg infusions of rituximab, and 61 patients were treated with two 1,000-mg infusions of rituximab. Highly sensitive flow cytometry was performed at 0, 2, 6, 14, and 26 weeks. European League Against Rheumatism (EULAR) response rates at 6 months were compared between patients with and those without complete depletion at each dose. RESULTS: The median B cell count was numerically higher at all time points following therapy in the 500 mg rituximab group. Twenty-five percent of patients in the 500 mg rituximab group had complete depletion at 2 weeks, compared with 49% of those in the 1,000 mg rituximab group. Complete depletion at 2 weeks after treatment with 500 mg rituximab was associated with lower baseline preplasma cell counts (P = 0.047). Most patients responded after either dose, but response was related to B cell depletion. Notably, in the 500 mg rituximab group all patients with complete depletion had a EULAR good response (P = 0.011). CONCLUSION: This pilot study suggests that the degree of B cell depletion, rather than the dose of rituximab, determines clinical response. It may be possible to predict which patients will respond to lower-dose rituximab, and this may allow more cost-effective treatment.

Challenges and solutions to recruitment of neonates and children having cardiac surgery into a study using a novel sampling device.

OBJECTIVE: To narratively describe the challenges and solutions required in delivering a non-commercial study of children undergoing cardiac surgery using a novel subcutaneous hormone collection device. RESULTS: The challenges faced by the research team are divided into those of conducting healthcare research in children and those specific to this study. Many of the issues of conducting healthcare research in children can and have been overcome by structural and institutional culture change-normalising and embedding research as part of good clinical care. The issues specific to insertion and maintenance of the novel collection device can be overcome by education and support of the clinical teams. The increased incentives and resources of commercial research may have overcome many of these.

A pre-registered, multi-lab non-replication of the action-sentence compatibility effect (ACE).

The Action-sentence Compatibility Effect (ACE) is a well-known demonstration of the role of motor activity in the comprehension of language. Participants are asked to make sensibility judgments on sentences by producing movements toward the body or away from the body. The ACE is the finding that movements are faster when the direction of the movement (e.g., toward) matches the direction of the action in the to-be-judged sentence (e.g., Art gave you the pen describes action toward you). We report on a pre-registered, multi-lab replication of one version of the ACE. The results show that none of the 18 labs involved in the study observed a reliable ACE, and that the meta-analytic estimate of the size of the ACE was essentially zero.

Ligament and bone pathologic abnormalities more frequent in neuropathic joint disease in comparison with degenerative arthritis of the foot and ankle: implications for understanding rapidly progressive joint degeneration.

OBJECTIVE: The variable disease progression of osteoarthritis (OA) and the basis for rapid joint deterioration in some subgroups of patients are poorly understood. To explore an anatomic basis for rapidly progressive OA, this observational study compared the magnetic resonance imaging (MRI) patterns of disease between patients with neuropathic joint disease (NJD) and patients with degenerative arthritis of the ankle and foot. METHODS: MR images of the foot and ankle of patients with early NJD (n = 7) and patients with OA (n = 15) were assessed. The anonomized MR images were dichotomously scored by a musculoskeletal radiologist for the presence of the following abnormalities per bone (of a total of 14 bones): cartilage defects, bone cysts, bone marrow edema, fractures, joint debris, joint effusions, tendinopathy, tendinitis, and ligament tears. RESULTS: Although the degree of cartilage damage and joint cyst formation was comparable between the groups, the degree of ligament tears, or change in MRI signal intensity in the ligaments, was significantly greater in patients with NJD compared with patients with OA (median of 3 tears versus 0, of 14 total bones; P < 0.01). Moreover, in patients with early NJD compared with patients with OA, there was a significantly greater degree of diffuse bone marrow edema (median of 6.5 tarsal bones versus 2 adjacent bones, of 14 total bones; P < 0.01), a greater number of bone fractures (median 4 versus 0; P < 0.01), and more frequent bone debris (median 4.5 versus 0; P = 0.013). CONCLUSION: This analysis of NJD in the foot and ankle shows the predominance of bone and ligament abnormalities in NJD compared with the pattern of involvement in OA. These findings highlight the importance of structures other than articular cartilage in OA of the ankle and foot, and suggest that rapid joint degeneration in NJD may be more ligamentogenic or osteogenic in nature.

Managing patients with resistant rheumatoid arthritis.

This article describes the management of patients with difficult to treat rheumatoid arthritis. Pharmacological treatment of patients using disease-modifying and biologic drugs is discussed. Clinical measures of disease activity are also described.

CUL5 is required for thalidomide-dependent inhibition of cellular proliferation.

Angiogenesis is essential for cancer metastasis, thus the discovery and characterization of molecules that inhibit this process is important. Thalidomide is a teratogenic drug which is known to inhibit angiogenesis and effectively inhibit cancer metastasis, yet the specific cellular targets for its effect are not well known. We discovered that CUL5 (previously identified as VACM-1), a scaffold protein in E3 ligase complexes, is involved in thalidomide-dependent inhibition of endothelial cell growth. Our results show that in human endothelial cells (HUVEC), thalidomide-dependent decrease in cell growth was associated with decreased nuclear localization of CUL5. In HUVEC transfected with anti-VACM-1 siRNA, thalidomide failed to decrease cell growth. Previously it was established that the antiproliferative effect of CUL5 is inhibited in rat endothelial cells (RAMEC) transfected with mutated CUL5 which is constitutively modified by NEDD8, a ubiquitin-like protein. In this study, the antiproliferative response to thalidomide was compromised in RAMEC expressing mutated CUL5. These results suggest that CUL5 protein is involved in the thalidomide-dependent regulation of cellular proliferation in vitro. Consequently, CUL5 may be an important part of the mechanism for thalidomide-dependent inhibition of cellular proliferation, as well as a novel biomarker for predicting a response to thalidomide for the treatment of disorders such as multiple myeloma and HIV infection.

Heterogeneity of Human Breast Stem and Progenitor Cells as Revealed by Transcriptional Profiling.

During development, the mammary gland undergoes extensive remodeling driven by stem cells. Breast cancers are also hierarchically organized and driven by cancer stem cells characterized by CD44+CD24low/- or aldehyde dehydrogenase (ALDH) expression. These markers identify mesenchymal and epithelial populations both capable of tumor initiation. Less is known about these populations in non-cancerous mammary glands. From RNA sequencing, ALDH+ and ALDH-CD44+CD24- human mammary cells have epithelial-like and mesenchymal-like characteristics, respectively, with some co-expressing ALDH+ and CD44+CD24- by flow cytometry. At the single-cell level, these cells have the greatest mammosphere-forming capacity and express high levels of stemness and epithelial-to-mesenchymal transition-associated genes including ID1, SOX2, TWIST1, and ZEB2. We further identify single ALDH+ cells with a hybrid epithelial/mesenchymal phenotype that express genes associated with aggressive triple-negative breast cancers. These results highlight single-cell analyses to characterize tissue heterogeneity, even in marker-enriched populations, and identify genes and pathways that define this heterogeneity.

Macrophages Enhance Migration in Inflammatory Breast Cancer Cells via RhoC GTPase Signaling.

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. All IBC patients have lymph node involvement and one-third of patients already have distant metastasis at diagnosis. This propensity for metastasis is a hallmark of IBC distinguishing it from less lethal non-inflammatory breast cancers (nIBC). Genetic profiling studies have been conducted to differentiate IBC from nIBC, but no IBC cancer-cell-specific gene signature has been identified. We hypothesized that a tumor-extrinsic factor, notably tumor-associated macrophages, promotes and contributes to IBC's extreme metastatic phenotype. To this end, we studied the effect of macrophage-conditioned media (MCM) on IBC. We show that two IBC cell lines are hyper-responsive to MCM as compared to normal-like breast and aggressive nIBC cell lines. We further interrogated IBC's hyper-responsiveness to MCM using a microfluidic migration device, which permits individual cell migration path tracing. We found the MCM "primes" the IBC cells' cellular machinery to become extremely migratory in response to a chemoattractant. We determined that interleukins -6, -8, and -10 within the MCM are sufficient to stimulate this enhanced IBC migration effect, and that the known metastatic oncogene, RhoC GTPase, is necessary for the enhanced migration response.

EGFR Tyrosine 845 Phosphorylation-Dependent Proliferation and Transformation of Breast Cancer Cells Require Activation of p38 MAPK.

Phosphorylation of epidermal growth factor receptor (EGFR) on tyrosine 845 by c-Src has been shown to be important for cell proliferation and migration in several model systems. This cross talk between EGFR and Src family kinases (SFKs) is one mechanism for resistance to EGFR inhibitors both in cell models and in the clinic. Here, we show that phosphorylation of tyrosine 845 on EGFR is required for proliferation and transformation using several cell models of breast cancer. Overexpression of EGFR-Y845F or treating cells with the SFK inhibitor dasatinib abrogated tyrosine 845 phosphorylation, yet had little to no effect on other EGFR phosphorylation sites or EGFR kinase activity. Abrogation of Y845 phosphorylation inhibited cell proliferation and transformation, even though extracellular signal-regulated kinase (ERK) and Akt remained active under these conditions. Importantly, cotransfection of mitogen-activated protein kinase (MAPK) kinase 3 and p38 MAPK restored cell proliferation in the absence of EGFR tyrosine 845 phosphorylation. Taken together, these data demonstrate a novel role for p38 MAPK signaling downstream of EGFR tyrosine 845 phosphorylation in the regulation of breast cancer cell proliferation and transformation and implicate SFK inhibitors as a potential therapeutic mechanism for overcoming EGFR tyrosine kinase inhibitor resistance in breast cancer.

Successful treatment of resistant pseudogout with anakinra.

We describe herein the case of a 63-year-old man with pseudogout affecting multiple joints that was resistant to treatment with allopurinol, steroids, and antiinflammatory drugs. Based on recent data on the molecular mechanism of pseudogout that demonstrated overproduction of interleukin-1 (IL-1), we treated the patient with anakinra, an IL-1 receptor antagonist. The patient responded to treatment with anakinra within 2 weeks, with resolution of the signs and symptoms of pseudogout and normalization of levels of inflammation markers.