TGF-beta signalling in bovine mammary gland involution and a comparative assessment of MAC-T and BME-UV1 cells as in vitro models for its study

The goal of the dairy industry is ultimately to increase lactation persistency, which is the length of time during which peak milk yield is sustained. Lactation persistency is determined by the balance of cell apoptosis and cell proliferation; when the balance is skewed toward the latter, this results in greater persistency. Thus, we can potentially increase milk production in dairy cows through manipulating apoptogenic and antiproliferative cellular signaling that occurs in the bovine mammary gland. Transforming growth factor beta 1 (TGFβ1) is an antiproliferative and apoptogenic cytokine that is upregulated during bovine mammary gland involution. Here, we discuss possible applications of TGFβ1 signaling for the purposes of increasing lactation persistency. We also compare the features of mammary alveolar cells expressing SV-40 large T antigen (MAC-T) and bovine mammary epithelial cells-clone UV1 (BME-UV1) cells, two extensively used bovine mammary epithelial cell lines, to assess their appropriateness for the study of TGFβ1 signaling. TGFβ1 induces apoptosis and arrests cell growth in BME-UV1 cells, and this was reported to involve suppression of the somatotropic axis. Conversely, there is no proof that exogenous TGFβ1 induces apoptosis of MAC-T cells. In addition to TGFβ1’s different effects on apoptosis in these cell lines, hormones and growth factors have distinct effects on TGFβ1 secretion and synthesis in MAC-T and BME-UV1 cells as well. MAC-T and BME-UV1 cells may behave differently in response to TGFβ1 due to their contrasting phenotypes; MAC-T cells have a profile indicative of both myoepithelial and luminal populations, while the BME-UV1 cells exclusively contain a luminal-like profile. Depending on the nature of the research question, the use of these cell lines as models to study TGFβ1 signaling should be carefully tailored to the questions asked

Clinical–pathologic significance of cancer stem cell marker expression in familial breast cancers

Human breast cancer cells with a CD44(+)/CD24(−/low) or ALDH1+ phenotype have been demonstrated to be enriched for cancer stem cells (CSCs) using in vitro and in vivo techniques. The aim of this study was to determine the association between CD44(+)/CD24(−/low) and ALDH1 expression with clinical–pathologic tumor characteristics, tumor molecular subtype, and survival in a well characterized collection of familial breast cancer cases. 364 familial breast cancers from the Ontario Familial Breast Cancer Registry (58 BRCA1-associated, 64 BRCA2-associated, and 242 familial non-BRCA1/2 cancers) were studied. Each tumor had a centralized pathology review performed. TMA sections of all tumors were analyzed for the expression of ER, PR, HER2, CK5, CK14, EGFR, CD44, CD24, and ALDH1. The Chi square test or Fisher’s exact test was used to analyze the marker associations with clinical–pathologic tumor variables, molecular subtype and genetic subtype. Analyses of the association of overall survival (OS) with marker status were conducted using Kaplan–Meier plots and log-rank tests. The CD44(+)/CD24(−/low) and ALDH1+ phenotypes were identified in 16% and 15% of the familial breast cancer cases, respectively, and associated with high-tumor grade, a high-mitotic count, and component features of the medullary type of breast cancer. CD44(+)/CD24(−/low) and ALDH1 expression in this series were further associated with the basal-like molecular subtype and the CD44(+)/CD24(−/low) phenotype was independently associated with BRCA1 mutational status. The currently accepted breast CSCs markers are present in a minority of familial breast cancers. Whereas the presence of these markers is correlated with several poor prognostic features and the basal-like subtype of breast cancer, they do not predict OS

Inhibition Of Epithelial-mesenchymal Transition And Metastasis By Combined Tgfbeta Knockdown And Metformin Treatment In A Canine Mammary Cancer Xenograft Model

Epithelial mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Transforming growth factor beta (TGF-β) is associated with this malignancy by having the ability to induce EMT. Metformin, has been shown to inhibit EMT in breast cancer cells. Based on this evidence we hypothesize that treatment with metformin and the silencing of TGF-β, inhibits the EMT in cancer cells. Canine metastatic mammary tumor cell line CF41 was stably transduced with a shRNA-lentivirus, reducing expression level of TGF-β1. This was combined with metformin treatment, to look at effects on cell migration and the expression of EMT markers. For in vivo study, unmodified or TGF-β1sh cells were injected in the inguinal region of nude athymic female mice followed by metformin treatment. The mice’s lungs were collected and metastatic nodules were subsequently assessed for EMT markers expression. The migration rate was lower in TGF-β1sh cells and when combined with metformin treatment. Metformin treatment reduced N-cadherin and increased E-cadherin expression in both CF41 and TGF-β1sh cells. Was demonstrated that metformin treatment reduced the number of lung metastases in animals bearing TGF-β1sh tumors. This paralleled a decreased N-cadherin and vimentin expression, and increased E-cadherin and claudin-7 expression in lung metastases. This study confirms the benefits of TGF-β1 silencing in addition to metformin as potential therapeutic agents for breast cancer patients, by blocking EMT process. To the best of our knowledge, we are the first to report metformin treatment in cells with TGF-β1 silencing and their effect on EMT. © 2017, Springer Science+Business Media New York.221274

Suppressive impact of metronomic chemotherapy using UFT and/or cyclophosphamide on mediators of breast cancer dissemination and invasion

Producción CientíficaMetronomic chemotherapy using the 5-FU prodrug uracil-tegafur (UFT) and cyclophosphamide (CTX) was previously shown to only modestly delay primary tumor growth, but nevertheless markedly suppressed the development of micro-metastasis in an orthotopic breast cancer xenograft model, using the metastatic variant of the MDA-MB-231 cell line, 231/LM2-4. Furthermore, a remarkable prolongation of survival, with no toxicity, was observed in a model of postsurgical advanced metastatic disease. A question that has remained unanswered is the seemingly selective anti-metastatic mechanisms of action responsible for this treatment. We assessed the in vivo effect of metronomic UFT, CTX or their combination, on vascular density, collagen deposition and c-Met (cell mediators or modulators of tumor cell invasion or dissemination) via histochemistry/immunohistochemistry of primary tumor sections. We also assessed the effect of continuous exposure to low and non-toxic doses of active drug metabolites 5-fluorouracil (5-FU), 4-hydroperoxycyclophosphamide (4-HC) or their combination, on 231/LM2-4 cell invasiveness in vitro. In the in vivo studies, a significant reduction in vascular density and p-Met[Y1003] levels was associated with UFT+CTX treatment. All treatments reduced intratumoral collagen deposition. In the in vitro studies, a significant reduction of collagen IV invasion by all treatments was observed. The 3D structures formed by 231/LM2-4 on Matrigel showed a predominantly Mass phenotype under treated conditions and Stellate phenotype in untreated cultures. Taken together, the results suggest the low-dose metronomic chemotherapy regimens tested can suppress several mediators of tumor invasiveness highlighting a new perspective for the anti-metastatic efficacy of metronomic chemotherapy.Canadian Institutes of Health Research (grant 364411

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

Dual EGFR and mTOR targeting in squamous cell carcinoma models, and development of early markers of efficacy

The epidermal growth factor receptor (EGFR) is a validated target in squamous cell carcinoma (SCC) of the head and neck. Most patients, however, do not respond or develop resistance to this agent. Mammalian target of rapamycin (mTOR) is involved in the pathogenesis of SCC of the head and neck (SCCHN). This study aimed to determine if targeting mTOR in combination with EGFR is effective in SCC, and to develop early pharmacodynamic markers of efficacy. Two SCC cell lines, one resistant (HEP2) and one of intermediate susceptibility (Detroit 562) to EGFR inhibitors, were xenografted in vivo and treated with an mTOR inhibitor (temsirolimus), an EGFR inhibitor (erlotinib) or a combination of both. Temsirolimus exerted superior growth arrest in both cell lines than erlotinib. The combined treatment resulted in synergistic antitumor effects in the Detroit 562 cell line. Immunohistochemical assessment of pharmacodynamic effects in fine-needle aspiration (FNA) biopsies early after treatment using phospho MAPK, Phospho-P70 and Ki67 as end points demonstrated pathway abrogation in the Detroit 562 tumours treated with the combination, the only group where regressions were seen. In conclusion, an mTOR inhibitor showed antitumor activity in EGFR-resistant SCC cell lines. Marked antitumor effects were associated with dual pathway inhibition, which were detected by early FNA biopsies

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

PTEN status in advanced colorectal cancer treated with cetuximab

BACKGROUND: Loss of phosphatase and tensin homologue deleted in chromosome 10 (PTEN) function in advanced colorectal cancer (CRC) may represent one of the resistance mechanisms to cetuximab by interfering with the epidermal growth factor receptor signal transduction pathway. METHODS: PTEN expression tested by indirect immunofluorescence was evaluated both on primary (n¼43) and on metastatic (n¼24) sites in CRC patients treated with cetuximab. RESULTS: The loss of PTEN expression tested on metastatic sites was negatively associated with response (100% progressive disease (PD) in PTEN-negative cases vs 30% PD in PTEN-positive cases; Po0.05), PFS (0.8 vs 8.2 months; Po0.001) and OS (2.9 vs 14.2 months; Po0.001). CONCLUSION: A potential role of PTEN in the anti-tumour activity of cetuximab could be hypothesised

Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of the HER inhibitors and cytotoxic drugs

Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 µM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2). Finally, of the TKIs, only treatment with afatinib, neratinib and dasatinib were able to reduce the migration of HER-2 overexpressing SKOV3 cells. We did not find any significant association between the expression of putative ovarian CSC marker, HER family members, c-MET, ALK, and IGF-IR and the response to the irreversible HER TKIs. Our results support the need for further investigations of the therapeutic potential of these irreversible HER family blockers in ovarian cancer, and the therapeutic potential of dasatinib when used in combination with the inhibitors of the HER family members in ovarian cancer