Collagen overlays can inhibit leptin and adiponectin secretion but not lipid accumulation in adipocytes

Background White adipose tissue (WAT) is essential for energy storage as well as being an active endocrine organ. The secretion of adipokines by adipocytes can affect whole body metabolism, appetite, and contribute to overall health. WAT is comprised of lipid-laden mature adipocytes, as well as immune cells, endothelial cells, pre-adipocytes, and adipose-derived stem cells. In addition, the presence of extracellular matrix (ECM) proteins in WAT can actively influence adipocyte differentiation, growth, and function. Type I collagen is an abundant fibrous ECM protein in WAT that is secreted by developing adipocytes. However, the extent and overall effect of Type I collagen on adipokine secretion in mature adipocytes when added exogenously has not been established. Methods We characterized the effects of Type I collagen overlays prepared using two different buffers on adipocyte physiology and function when added at different times during differentiation. In addition, we compared the effect of collagen overlays when adipocytes were cultured on two different tissue culture plastics that have different adherent capabilities. Triglyceride accumulation was analyzed to measure adipocyte physiology, and leptin and adiponectin secretion was determined to analyze effects on adipokine secretion. Results We found that collagen overlays, particularly when added during the early differentiation stage, impaired adipokine secretion from mature adipocytes. Collagen prepared using PBS had a greater suppression of leptin than adiponectin while collagen prepared using HANKS buffer suppressed the secretion of both adipokines. The use of CellBind plates further suppressed leptin secretion. Triglyceride accumulation was not substantially impacted with any of the collagen overlays. Discussion Adipokine secretion can be selectively altered by collagen overlays. Thus, it is feasible to selectively manipulate the secretion of adipokines by adipocytes in vitro by altering the composition or timing of collagen overlays. The use of this technique could be applied to studies of adipokine function and secretion in vitro as well as having potential therapeutic implications to specifically alter adipocyte functionality in vivo

Identification of lysyl oxidase as an adipocyte-secreted mediator that promotes a partial mesenchymal-to-epithelial transition in MDA-MB-231 cells

Aim: Breast cancer (BC) is the most common cancer in women worldwide, where adiposity has been linked to BC morbidity. In general, obese premenopausal women diagnosed with triple-negative BC (TNBC) tend to have larger tumours with more metastases, particularly to the bone marrow, and worse prognosis. Previous work using a 3-dimensional (3D) co-culture system consisting of TNBC cells, adipocytes and the laminin-rich extracellular matrix (ECM) trademarked as Matrigel, demonstrated that adipocytes and adipocyte-derived conditioned media (CM) caused a partial mesenchymal-to-epithelial transition (MET). Given that MET has been associated with secondary tumour formation, this study sought to identify molecular mediators responsible for this phenotypic change. Methods: Adipocytes were cultured with and without Matrigel, where semi-quantitative proteomics was used to identify proteins whose presence in the CM was induced or enhanced by Matrigel, which were referred to as adipocyte-secreted ECM-induced proteins (AEPs). The AEPs identified were assessed for association with prognosis in published proteomic datasets and prior literature. Of these, 4 were evaluated by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), followed by a functional and MET marker analysis of 1 AEP on MDA-MB-231 cells grown on Matrigel or as monolayers. Results: The 4 AEPs showed a positive correlation between protein expression and poor prognosis. RT-qPCR analysis reported no significant change in AEPs mRNA expression. However, lysyl oxidase (LOX) was increased in CM of ECM-exposed adipocytes. Recombinant LOX (rLOX) caused the mesenchymal MDA-MB-231 TNBC cells to form less branched 3D structures and reduced the expression of vimentin. Conclusions: The data suggest that adipocyte-secreted LOX changes the mesenchymal phenotype of BC cells in a manner that could promote secondary tumour formation, particularly at sites high in adipocytes such as the bone marrow. Future efforts should focus on determining whether targeting LOX could reduce BC metastasis in obese individuals

The function and therapeutic targeting of anaplastic lymphoma kinase (ALK) in non-small cell lung cancer (NSCLC)

Abstract Lung cancer is the leading cause of death by cancer in North America. A decade ago, genomic rearrangements in the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase were identified in a subset of non-small cell lung carcinoma (NSCLC) patients. Soon after, crizotinib, a small molecule ATP-competitive ALK inhibitor was proven to be more effective than chemotherapy in ALK-positive NSCLC patients. Crizotinib and two other ATP-competitive ALK inhibitors, ceritinib and alectinib, are approved for use as a first-line therapy in these patients, where ALK rearrangement is currently diagnosed by immunohistochemistry and in situ hybridization. The clinical success of these three ALK inhibitors has led to the development of next-generation ALK inhibitors with even greater potency and selectivity. However, patients inevitably develop resistance to ALK inhibitors leading to tumor relapse that commonly manifests in the form of brain metastasis. Several new approaches aim to overcome the various mechanisms of resistance that develop in ALK-positive NSCLC including the knowledge-based alternate and successive use of different ALK inhibitors, as well as combined therapies targeting ALK plus alternative signaling pathways. Key issues to resolve for the optimal implementation of established and emerging treatment modalities for ALK-rearranged NSCLC therapy include the high cost of the targeted inhibitors and the potential of exacerbated toxicities with combination therapies

Signalling by Transforming Growth Factor Beta Isoforms in Wound Healing and Tissue Regeneration

Transforming growth factor beta (TGFβ) signalling is essential for wound healing, including both non-specific scar formation and tissue-specific regeneration. Specific TGFβ isoforms and downstream mediators of canonical and non-canonical signalling play different roles in each of these processes. Here we review the role of TGFβ signalling during tissue repair, with a particular focus on the prototypic isoforms TGFβ1, TGFβ2, and TGFβ3. We begin by introducing TGFβ signalling and then discuss the role of these growth factors and their key downstream signalling mediators in determining the balance between scar formation and tissue regeneration. Next we discuss examples of the pleiotropic roles of TGFβ ligands during cutaneous wound healing and blastema-mediated regeneration, and how inhibition of the canonical signalling pathway (using small molecule inhibitors) blocks regeneration. Finally, we review various TGFβ-targeting therapeutic strategies that hold promise for enhancing tissue repair

Proteasome Inhibitors and Their Potential Applicability in Osteosarcoma Treatment

Osteosarcoma (OS) is the most common type of bone cancer, with ~30% of patients developing secondary/metastatic tumors. The molecular complexity of tumor metastasis and the lack of effective therapies for OS has cultivated interest in exploiting the proteasome as a molecular target for anti-cancer therapy. As our understanding towards the behavior of malignant cells expands, it is evident that cancerous cells display a greater reliance on the proteasome to maintain homeostasis and sustain efficient biological activities. This led to the development and approval of first- and second-generation proteasome inhibitors (PIs), which have improved outcomes for patients with multiple myeloma and mantle cell lymphoma. Researchers have since postulated the therapeutic potential of PIs for the treatment of OS. As such, this review aims to summarize the biological effects and latest findings from clinical trials investigating PI-based treatments for OS. Integrating PIs into current treatment regimens may better outcomes for patients diagnosed with OS

Adipose Tissue from Lean and Obese Mice Induces a Mesenchymal to Epithelial Transition-Like Effect in Triple Negative Breast Cancers Cells Grown in 3-Dimensional Culture

Breast cancer is the second leading cause of cancer-related mortality among women globally with obesity being one risk factor. Obese breast cancer patients have at least a 30% increased risk of death from breast cancer compared to non-obese breast cancer patients because they present with larger tumors and generally have increased rates of metastasis. Moreover, obese breast cancer patients respond more poorly to treatment compared to non-obese patients, particularly pre-menopausal women diagnosed with triple negative breast cancer (TNBC). To help understand the molecular mechanisms underlying the increased metastasis associated with obesity, we previously established a three-dimensional culture system that permits the co-culture of adipocytes and TNBC cells in a manner that mimics an in vivo milieu. Using this system, we demonstrate that white adipose tissue from both lean and obese mice can induce a partial mesenchymal-to-epithelial transition (MET). Triple negative breast cancer cells adopt an epithelial morphology and have an increased expression of some epithelial markers, but they maintain the expression of mesenchymal markers, furnishing the breast cancer cells with hybrid properties that are associated with more aggressive tumors. Thus, these data suggest that adipose tissue has the potential to promote secondary tumor formation in lean and obese women. Further work is needed to determine if targeting the partial MET induced by adipose tissue could reduce metastasis

Par6 is an essential mediator of apoptotic response to transforming growth factor beta in NMuMG immortalized mammary cells

Abstract Background We previously observed that the TGFbeta-Par6 pathway mediates loss of polarity and apoptosis in NMuMG cells. Here we investigate the contribution of Par6 versus TGFbeta receptor I activation to TGFbeta-induced apoptosis in association with changes in apico-basal polarity. We focus on the effect of Par6 activation on alpha6beta4 integrin expression and localization, and Nuclear Factor-kappaB (p65/RelA) activation, previously shown to mediate polarity-dependent cell survival. Methods Using immunoblotting and/or immunofluorescence we investigated the effect of TGFbeta1 on apoptosis, alpha6, beta4 and beta1 integrin expression/localization, and p65/RelA phosphorylation/localization in monolayer and three-dimensional (3D) cultures of NMuMG cells with an overactive or inactive Par6 pathway. Results were quantified by band densitometry or as percent of 3D structures displaying a phenotype. Differences among means were compared by two-way ANOVA. Results Blocking Par6 activation inhibits TGFbeta-induced apoptosis. Par6 overactivation enhances TGFbeta-induced apoptosis, notably after 6-day exposure to TGFbeta (p < 0.001), a time when parental NMuMG cells no longer respond to TGFbeta apoptotic stimuli. 48-hour TGFbeta treatment reduced beta4 integrin levels in NMuMG monolayers and significantly reduced the basal localization of alpha6 (p < 0.001) and beta4 (p < 0.001) integrin in NMuMG 3D structures, which was dependent on both Par6 and TGFbeta receptor I activation and paralleled apoptotic response. After 6-day exposure to TGFbeta, Par6-dependent changes to beta4 integrin were no longer apparent, but there was reduced phosphorylation of p65/RelA (p < 0.001) only in Par6 overexpressing cells. Differences in p65/RelA localization were not observed among the different cell lines after 48-hour TGFbeta exposure. Conclusions Par6 and TGFbeta receptor I activation are both necessary for TGFbeta-induced apoptosis in NMuMG cells. Importantly, Par6 overexpression enhances the sensitivity of NMuMG to TGFbeta-induced apoptosis, notably upon prolonged exposure to this growth factor, when NMuMG parental cells are usually apoptosis-resistant. Thus, endogenous Par6 level might be important in determining whether TGFbeta will function as either a pro-apoptotic or pro-survival factor in breast cancer, and potentially aid in predicting patient’s prognosis and therapy response

Strategies for Delaying or Treating In vivo Acquired Resistance to Trastuzumab in Human Breast Cancer Xenografts

Purpose: Acquired resistance to trastuzumab (Herceptin) is common in patients whose breast cancers show an initial response to the drug. The basis of this acquired resistance is unknown, hampering strategies to delay or treat such acquired resistance, due in part to the relative lack of appropriate in vivotumorigenic models. Experimental Design: We derived an erbB-2–positive variant called 231-H2N, obtained by gene transfection from the highly tumorigenic erbB-2/HER2–negative human breast cancer cell line, MDA-MB-231. Unlike MDA-MB-231, the 231-H2N variants was sensitive to trastuzumab both in vitro and especially in vivo, thus allowing selection of variant resistant to drug treatment in the latter situation after showing an initial response. Results: The growth of established orthotopic tumors in severe combined immunodeficient mice was blocked for 1 month by trastuzumab, after which rapid growth resumed. These relapsing tumors were found to maintain resistance to trastuzumab, both in vitro and in vivo. We evaluated various therapeutic strategies for two purposes: (a) to delay such tumor relapses or (b) to treat acquired trastuzumab resistance once it has occurred. With respect to the former, a daily oral low-dose metronomic cyclophosphamide regimen was found to be particularly effective. With respect to the latter, an anti–epidermal growth factor receptor antibody (cetuximab) was effective as was the anti–vascular endothelial growth factor (anti-VEGF) antibody bevacizumab, which was likely related to elevated levels of VEGF detected in trastuzumab-resistant tumors. Conclusions: Our results provide a possible additional rationale for combined biological therapy using drugs that target both erbB-2/HER2 and VEGF and also suggest the potential value of combining less toxic metronomic chemotherapy regimens not only with targeted antiangiogenic agents but also with other types of drug such as trastuzumab

Proteomic Assessment of Extracellular Vesicles from Canine Tissue Explants as a Pipeline to Identify Molecular Targets in Osteosarcoma: PSMD14/Rpn11 as a Proof of Principle

Osteosarcoma (OS) is a highly malignant bone tumour that has seen little improvement in treatment modalities in the past 30 years. Understanding what molecules contribute to OS biology could aid in the discovery of novel therapies. Extracellular vesicles (EVs) serve as a mode of cell-to-cell communication and have the potential to uncover novel protein signatures. In our research, we developed a novel pipeline to isolate, characterize, and profile EVs from normal bone and osteosarcoma tissue explants from canine OS patients. Proteomic analysis of vesicle preparations revealed a protein signature related to protein metabolism. One molecule of interest, PSMD14/Rpn11, was explored further given its prognostic potential in human and canine OS, and its targetability with the drug capzimin. In vitro experiments demonstrated that capzimin induces apoptosis and reduces clonogenic survival, proliferation, and migration in two metastatic canine OS cell lines. Capzimin also reduces the viability of metastatic human OS cells cultured under 3D conditions that mimic the growth of OS cells at secondary sites. This unique pipeline can improve our understanding of OS biology and identify new prognostic markers and molecular targets for both canine and human OS patients