CSF-1R as an inhibitor of apoptosis and promoter of proliferation, migration and invasion of canine mammary cancer cells

BACKGROUND: Tumor-associated macrophages (TAMs) have high impact on the cancer development because they can facilitate matrix invasion, angiogenesis, and tumor cell motility. It gives cancer cells the capacity to invade normal tissues and metastasize. The signaling of colony-stimulating factor-1 receptor (CSF-1R) which is an important regulator of proliferation and differentiation of monocytes and macrophages regulates most of the tissue macrophages. However, CSF-1R is expressed also in breast epithelial tissue during some physiological stages i.g.: pregnancy and lactation. Its expression has been also detected in various cancers. Our previous study has showed the expression of CSF-1R in all examined canine mammary tumors. Moreover, it strongly correlated with grade of malignancy and ability to metastasis. This study was therefore designed to characterize the role of CSF-1R in canine mammary cancer cells proliferation, apoptosis, migration, and invasion. As far as we know, the study presented hereby is a pioneering experiment in this field of veterinary medicine. RESULTS: We showed that csf-1r silencing significantly increased apoptosis (Annexin V test), decreased proliferation (measured as Ki67 expression) and decreased migration (“wound healing” assay) of canine mammary cancer cells. Treatment of these cells with CSF-1 caused opposite effect. Moreover, csf-1r knock-down changed growth characteristics of highly invasive cell lines on Matrigel matrix, and significantly decreased the ability of these cells to invade matrix. CSF-1 treatment increased invasion of cancer cells. CONCLUSION: The evidence of the expression and functional role of the CSF-1R in canine mammary cancer cells indicate that CSF-1R targeting may be a good therapeutic approach

Migrastatin analogues inhibit canine mammary cancer cell migration and invasion.

BACKGROUND:Cancer spread to other organs is the main cause of death of oncological patients. Migration of cancer cells from a primary tumour is the crucial step in the complex process of metastasis, therefore blocking this process is currently the main treatment strategy. Metastasis inhibitors derived from natural products, such as, migrastatin, are very promising anticancer agents. Thus, the aim of our study was to investigate the effect of six migrastatin analogues (MGSTA-1 to 6) on migration and invasion of canine mammary adenocarcinoma cell lines isolated from primary tumours and their metastases to the lungs. Canine mammary tumours constitute a valuable tool for studying multiple aspect of human cancer. RESULTS:OUR RESULTS SHOWED THAT TWO OF SIX FULLY SYNTHETIC ANALOGUES OF MIGRASTATIN: MGSTA-5 and MGSTA-6 were potent inhibitors of canine mammary cancer cells migration and invasion. These data were obtained using the wound healing test, as well as trans-well migration and invasion assays. Furthermore, the treatment of cancer cells with the most effective compound (MGSTA-6) disturbed binding between filamentous F-actin and fascin1. Confocal microscopy analyses revealed that treatment with MGSTA-6 increased the presence of unbound fascin1 and reduced co-localization of F-actin and fascin1 in canine cancer cells. Most likely, actin filaments were not cross-linked by fascin1 and did not generate the typical filopodial architecture of actin filaments in response to the activity of MGSTA-6. Thus, administration of MGSTA-6 results in decreased formation of filopodia protrusions and stress fibres in canine mammary cancer cells, causing inhibition of cancer migration and invasion. CONCLUSION:Two synthetic migrastatin analogues (MGSTA-5 and MGSTA-6) were shown to be promising compounds for inhibition of cancer metastasis. They may have beneficial therapeutic effects in cancer therapy in dogs, especially in combination with other anticancer drugs. However, further in vivo studies are required to verify this hypothesis

Migrastatin analogues inhibit canine mammary cancer cell migration and invasion

Background: Cancer spread to other organs is the main cause of death of oncological patients. Migration of cancer cells from a primary tumour is the crucial step in the complex process of metastasis, therefore blocking this process is currently the main treatment strategy. Metastasis inhibitors derived from natural products, such as, migrastatin, are very promising anticancer agents. Thus, the aim of our study was to investigate the effect of six migrastatin analogues (MGSTA-1 to 6) on migration and invasion of canine mammary adenocarcinoma cell lines isolated from primary tumours and their metastases to the lungs. Canine mammary tumours constitute a valuable tool for studying multiple aspect of human cancer. Results: Our results showed that two of six fully synthetic analogues of migrastatin: MGSTA-5 and MGSTA-6 were potent inhibitors of canine mammary cancer cells migration and invasion. These data were obtained using the wound healing test, as well as trans-well migration and invasion assays. Furthermore, the treatment of cancer cells with the most effective compound (MGSTA-6) disturbed binding between filamentous F-actin and fascin1. Confocal microscopy analyses revealed that treatment with MGSTA-6 increased the presence of unbound fascin1 and reduced co-localization of F-actin and fascin1 in canine cancer cells. Most likely, actin filaments were not cross-linked by fascin1 and did not generate the typical filopodial architecture of actin filaments in response to the activity of MGSTA-6. Thus, administration of MGSTA-6 results in decreased formation of filopodia protrusions and stress fibres in canine mammary cancer cells, causing inhibition of cancer migration and invasion. Conclusion: Two synthetic migrastatin analogues (MGSTA-5 and MGSTA-6) were shown to be promising compounds for inhibition of cancer metastasis. They may have beneficial therapeutic effects in cancer therapy in dogs, especially in combination with other anticancer drugs. However, further in vivo studies are required to verify this hypothesis

Structures of migrastatin MGST-7 and tested migrastatin analogues MGSTA-1 to 6 and MGSTA-8.

<p>Structures of migrastatin MGST-7 and tested migrastatin analogues MGSTA-1 to 6 and MGSTA-8.</p

The effect of MGSTA-5 and MGSTA-6 on migration of canine mammary carcinoma cells evaluated by trans-well migration assay

<p>(A) Quantification of migration of examined cell lines (CMT-W1, CMT-W1M, CMT-W2, CMT-W2M) is presented as Relative Florescent Units (RFU) obtained using florescent plate reader Infinite 200 PRO Tecan™ (Ex. 485, Em. 530). The treatment of cancer cells with MGSTA-5 and MGSTA-6 for 20 hrs at the concentration of 100 µM decreased the migration through membrane of CMT-W1, and CMT-W1M cells and almost completely abolished migration of CMT-W2 cells. In CMT-W2M cell line those compounds were ineffective. The experiment was performed three times. The statistical analysis was done using Prism version 5.00 software (GraphPad Software, USA). One-way ANOVA followed by Tukey HSD post-hoc test and unpaired t-test were applied. p < 0.05 was marked as *, p < 0.001 was marked as ***. (B) Micrographs of the migrated CMT-W1, CMT-W1M and CMT-W2 cells taken with Olympus microscopy BX60 at x4 magnification.</p

Growth characteristics of CMT-W1, CMT-W2, CMT-W1M and CMT-W2M cell lines cultured on the Matrigel

<p>Matrix. Cell were incubated with MGSTA-6 or grown in control conditions for 24 hrs on 8-chamber culture slides coated with Matrigel™ Matrix (BD Biosciences, USA). In control condition cells presented branched shape, with numerous filopodia, typical for those invasive cells growing on Matrigel. The CMT-W1, CMT-W1M and CMT-W2 cells formed linear bindings, which created polygonal structures. The CMT-W2M cells formed lower amount of branches and did not created such structures. Administration of MGSTA-6 caused very potent inhibition of branches formation in CMT-W1, CMT-W1M and CMT-W2 cancer cell lines and decreased the amount of branches in CMT-W2M cell line. The round bands, packets of cells were observed in all fields of view after MGSTA-6 treatment. The micrographs were obtained with phase-contrast microscope at magnification of x4 (IX 70 Olympus Optical Co., Germany).</p

Wound healing assay of canine mammary carcinoma cells in control conditions and after treatment with six migrastatin analogues (MGSTA-1 to 6).

<p>Quantification of migration of CMT-W1 (A), CMT-W2 (B) CMT-W1M (C) cell lines are presented as percentages of scratch closure and were calculated as follows: % of scratch closure = a-b/a, where (a) is a distance between edges of the wound, and (b) is the distance which remained cell-free during cell migration to close the scratch. Photographs of cells invading the scratch were taken at the starting point and after 4, 6, 8, and 24 hrs using phase contrast microscopy (IX 70 Olympus Optical Co., Germany). Administration of MGSTA-5 and MGSTA-6 (100 µM) decreased cells motility in CMT-W1, CMT-W2 and CMT-W1M cell lines. The MGSTA-2 treatment decreased the cells motility only in the CMT-W2 cell line. Other migrastatin analogues do not affect migration ability of examined cell lines. (D) Representative images of scratch closure in control conditions and after MGSTA-5 and MGSTA-6 treatment (100 µM) at the 8^th hr after scratching in CMT-W1, CMT-W2 and CMT-W1M cell lines; images were taken using lens with 4x magnification and analysed using a computer-assisted image analyser (Olympus Microimage™ Image Analysis, software version 5.0 for Windows, USA). The experiment was performed three times. Two-way ANOVA and Bonferroni post-hoc tests were applied. p < 0.05 was marked as * and p < 0.001 was marked as ***.</p

Synthesis of migrastatin-core analogues.

<p>Reagents and conditions: (a) 5-hexenoic acid, Ph ₃P, DIAD, PhCH₃, rt, 3h, 69%; (b) Grubbs 2^nd, PhCH₃, reflux, 25 min, 68%; (c) HF. Py, THF, rt, 18h, 61%; (d) CBr₄, Ph ₃P polymer-bound, CH₂Cl_2, rt, 50 min; (e) <b>12</b>, DBU, PhCH₃ then <b>11</b>, rt, 1.5h; (f) Na/Hg, MeOH, rt, 3h, 51% over three steps; (g) Grubbs 2^nd, PhCH₃, reflux, 15 min, 99%; (h) HF. Py, THF, rt, 38h, 84%; (i) Lawesson reagent, CH₂Cl₂, mw, 100 °C, 10 min; (j) <b>9</b>, Ph ₃P, DIAD, PhCH₃, rt, 15h, 42%; (k) Grubbs 2^nd, PhCH₃, reflux, 15 min, 88%; (l) HF. Py, THF, rt, 18h, 85%; (m) TMSCl, LHMDS, THF, 0 °C, 2h; (n) Pd(OAc)₂, CH₃CN, rt, 3h, 64% over two steps; (o) HF. Py, THF, rt, 18h, 90%.</p

Macrophages Mediate a Switch between Canonical and Non-Canonical Wnt Pathways in Canine Mammary Tumors

<div><p>Objective</p><p>According to the current hypothesis, tumor-associated macrophages (TAMs) are “corrupted” by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell–TAM interactions are complicated and controversial we aimed to better define this phenomenon.</p><p>Methods and Results</p><p>Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial–mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs.</p><p>Conclusions</p><p>We demonstrated that TAMs mediate a “switch” between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis.</p><p>Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being “corrupted” by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis.</p><p>These data challenge the conventional understanding of TAM–cancer cell interactions.</p></div

Growth characteristic on the Matrigel matrix.

<p>Phase contrast micrographs of CMT-U27, CMT-U309, and P114 cells grown on the Matrigel matrix for 72 h under control conditions or in macrophage-conditioned medium. Control neoplastic cells formed colonies, whereas those treated with macrophage-conditioned medium invaded the Matrigel matrix and formed branches.</p