Disintegrin-like Protein Strategy to Inhibit Aggressive Triple-Negative Breast Cancer.

Venoms are a rich source of bioactive compounds, and among them is leberagin-C (Leb-C), a disintegrin-like protein derived from the venom of Macrovipera lebetina transmediterrannea snakes. Leb-C has shown promising inhibitory effects on platelet aggregation. Previous studies have demonstrated that this SECD protein specifically targets α5ß1, αvß3, and αvß6 integrins through a mimic mechanism of RGD disintegrins. In our current study, we focused on exploring the potential effects of Leb-C on metastatic breast cancer. Our findings revealed that Leb-C disrupted the adhesion, migration, and invasion capabilities of MDA-MB-231 breast cancer cells and its highly metastatic D3H2LN sub-population. Additionally, we observed significant suppression of adhesion, migration, and invasion of human umbilical vein endothelial cells (HUVECs). Furthermore, Leb-C demonstrated a strong inhibitory effect on fibroblast-growth-factor-2-induced proliferation of HUVEC. We conducted in vivo experiments using nude mice and found that treatment with 2 µM of Leb-C resulted in a remarkable 73% reduction in D3H2LN xenograft tumor size. Additionally, quantification of intratumor microvessels revealed a 50% reduction in tumor angiogenesis in xenograft after 21 days of twice-weekly treatment with 2 µM of Leb-C. Collectively, these findings suggest the potential utility of this disintegrin-like protein for inhibiting aggressive and resistant metastatic breast cancer.

Drug resistance­related sunitinib sequestration in autophagolysosomes of endothelial cells.

Our previous study demonstrated that the tyrosine kinase receptor inhibitor sunitinib induces acquired drug resistance in endothelial cells. The present study explored the role of lysosomal sequestration of sunitinib in the acquisition of drug resistance in human microcapillary endothelial HMEC­1 cells. Resistance was induced by escalating concentrations of sunitinib and a shift in IC50 from 12.8 to >20 µM was detected. The results of time­lapse fluorescence microscopy illustrated an instantaneous emergence of fluorescent vesicles in living cells once sunitinib was added. Most of these vesicles emerged in the juxtanuclear area, and exhibited the characteristics of growing autophagosomes and lysosomes. The vesicles were identified as autophagosomes and lysosomes because they co­located with the lysosomal tracers Lyso­ER and Lyso­NIR, and the protein markers lysosomal­associated membrane protein 1 (LAMP­1) and microtubule­associated protein 1A/1B­light chain 3 (LC3). The results of western blotting demonstrated that sunitinib induced upregulation of LAMP­1 and LC3­II, and downregulation of sequestosome 1/p62, indicating the activation of autophagy. Bafilomycin A1, which suppresses lysosomal acidification, completely blocked sunitinib sequestration; however, chloroquine, which blocks lysosomal fusion with autophagosomes, exhibited no effect. Notably, bafilomycin A1 and chloroquine significantly counterbalanced HMEC­1 drug­resistance. These results provided evidence for autophagy­flux­associated sunitinib lysosomal sequestration in endothelial cells, leading to isolation of the drug from the cytoplasm; a key process involved in the development of drug resistance during antiangiogenic therapy. These data supported the notion that inhibiting autophagy may be a potential strategy to prevent drug sequestration and resistance to antiangiogenic therapy.

Pulsed-laser irradiation of multifunctional gold nanoshells to overcome trastuzumab resistance in HER2-overexpressing breast cancer.

BACKGROUND: HER2-overexpressing metastatic breast cancers are challenging practice in oncology when they become resistant to anti-HER2 therapies such as trastuzumab. In these clinical situations, HER2-overexpression persists in metastatic localizations, and can thus be used for active targeting using innovative therapeutic approaches. Functionalized gold nanoparticles with anti-HER2 antibody can be stimulated by near-infrared light to induce hyperthermia. METHODS: Here, hybrid anti-HER2 gold nanoshells were engineered for photothermal therapy to overcome trastuzumab resistance in HER2-overexpressing breast cancer xenografts. RESULTS: When gold nanoshells were administered in HER2-tumor xenografts, no toxicity was observed. A detailed pharmacokinetic study showed a time-dependent accumulation of gold nanoshells within the tumors, significantly greater with functionalized gold nanoshells at 72 h. This enabled us to optimize the treatment protocol and irradiate the mice when the anti-HER2 gold nanoshells had accumulated most in the tumors. After weekly injections of anti-HER2 gold nanoshells, and repeated irradiations with a femtosecond-pulsed laser over four weeks, tumor growth was significantly inhibited. Detailed tissue microscopic analyses showed that the tumor growth inhibition was due to an anti-angiogenic effect, coherent with a preferential distribution of the nanoshells in tumor microvessels. We also showed a direct tumor cell effect with apoptosis and inhibition of proliferation, coherent with an immune-mediated targeting of tumor cells by anti-HER2 nanoshells. CONCLUSION: This preclinical study thus supports the use of anti-HER2 gold nanoshells and photothermal therapy to overcome trastuzumab resistance in HER2-overexpressing breast cancer.

Targeting Cancer Stem Cells to Overcome Chemoresistance.

Cancers are heterogeneous at the cell level, and the mechanisms leading to cancer heterogeneity could be clonal evolution or cancer stem cells. Cancer stem cells are resistant to most anti-cancer treatments and could be preferential targets to reverse this resistance, either targeting stemness pathways or cancer stem cell surface markers. Gold nanoparticles have emerged as innovative tools, particularly for photo-thermal therapy since they can be excited by laser to induce hyperthermia. Gold nanoparticles can be functionalized with antibodies to specifically target cancer stem cells. Preclinical studies using photo-thermal therapy have demonstrated the feasibility of targeting chemo-resistant cancer cells to reverse clinical chemoresistance. Here, we review the data linking cancer stem cells and chemoresistance and discuss the way to target them to reverse resistance. We particularly focus on the use of functionalized gold nanoparticles in the treatment of chemo-resistant metastatic cancers.

Primary mediastinal large B-cell lymphoma: transcriptional regulation by miR-92a through FOXP1 targeting.

BACKGROUND: Primary mediastinal large B-cell lymphoma (PMBL) shares pathological features with diffuse large B-cell lymphoma (DLBCL), and molecular features with classical Hodgkin lymphoma (cHL). The miR-17~92 oncogenic cluster, located at chromosome 13q31, is a region that is amplified in DLBCL. METHODS: Here we compared the expression of each member of the miR-17~92 oncogenic cluster in samples from 40 PMBL patients versus 20 DLBCL and 20 cHL patients, and studied the target genes linked to deregulated miRNA in PMBL. RESULTS: We found a higher level of miR-92a in PMBL than in DLBCL, but not in cHL. A combination of in silico prediction and transcriptomic analyses enabled us to identify FOXP1 as a main miR-92a target gene in PMBL, a result so far not established. This was confirmed by 3'UTR, and RNA and protein expressions in transduced cell lines. In vivo studies using the transduced cell lines in mice enabled us to demonstrate a tumor suppressor effect of miR-92a and an oncogenic effect of FOXP1.A higher expression of miR-92a and the down-regulation of FOXP1 mRNA and protein expression were also found in human samples of PMBL, while miR-92a expression was low and FOXP1 was high in DLBCL. CONCLUSIONS: We concluded to a post-transcriptional regulation by miR-92a through FOXP1 targeting in PMBL, with a clinico-pathological relevance for better characterisation of PMBL.

[Resistance to anti-angiogenic therapy: a clinical and scientific current issue]. / La résistance aux traitements antiangiogéniques - Une actualité clinique et scientifique.

Although the use of anti-angiogenic agents has been considered a promising strategy to block tumor growth and improve the bioavailability of drugs into the tumor, the use of most of them in clinical trials is limited. The development of resistance to some anti-angiogenic agents and their high toxicity are currently under investigations. However, the approach is still valid since this therapeutic tool has lengthened survival of patients with colon, breast, kidney, lungs and liver cancers. The identification of biomarkers in response to this family of drugs is an important area of investigation.

MDA-MB-231 breast cancer cells overexpressing single VEGF isoforms display distinct colonisation characteristics.

BACKGROUND: Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that has important roles in angiogenesis. Our knowledge of the significance of VEGF isoforms in human cancer remains incomplete. METHODS: Bioluminescence imaging and transcriptomic analysis were used to study the colonisation capacity of the human breast cancer cells MDA-MB-231 controlling or overexpressing the VEGF165 or VEGF189 isoform (named cV-B, V165-B and V189-B, respectively) in nude mice. RESULTS: When injected into the bloodstream, V189-B cells induced less metastasis in the lungs and bone than V165-B and cV-B control cells, consistent with longer survival of these mice and delay in tumour uptake in the mice injected with a V189-B clone. Histological analysis confirmed that there were less αSMA-positive cells in the lungs of the mice injected with V189-B. In vitro V189-B cells decreased both cell invasion and survival. Using transcriptomic analysis, we identified a subset of 18 genes expressed differentially between V189 and V165 cell lines and in 120 human breast tumours. V165 was associated with poor prognosis, whereas V189 was not, suggesting a complex regulation by VEGF isoforms. Our results showed a negative correlation between the expression pattern of VEGF189 and the levels of expression of seven genes that influence metastasis. CONCLUSION: Our findings provide the first evidence that VEGF isoforms have different effects on breast cancer cell line colonisation in vivo.

Cross-drug resistance to sunitinib induced by doxorubicin in endothelial cells.

Multiple drug resistance remains an unsolved problem in cancer therapy. A previous study has demonstrated that the chemotherapeutic drug doxorubicin (Dox) induced upregulation of P-glycoprotein in endothelial cells, resulting in a 20-fold increase in drug resistance and reduced efficiency of doxorubicin treatment in a mouse tumor model. In the present study, the cross-resistance and sensitivity of HMECd1 and HMECd2 established cell lines to anti-angiogenic drugs, particularly sunitinib, was explored. The results revealed that Dox treatment induced a significant increase in the breast cancer resistance protein (ABCG2) gene transcription and protein expression. This increase gave rise to a 4- to 5-fold increase in the half maximal inhibitory concentration of the HMECd1 and HMECd2 cells in response to sunitinib treatment in vitro. Functionally, the role of ABCG2 in the resistance to sunitinib was confirmed by the use of the ABCG2 inhibitors fumitremorgin C and diethylstilbestrol, which blocked cell resistance. The present study indicates that endothelial cells exhibit cross-resistance between cytotoxic drugs and anti-angiogenic drugs. This suggests that multiple drug resistance induced by chemotherapy in endothelial cells may affect the efficiency of anti-angiogenic drugs.

Bisphosphonate prodrugs: synthesis and biological evaluation in HuH7 hepatocarcinoma cells.

We investigated the biological effects of new synthesized bisphosphonates (BPs) on HuH7 hepatocarcinoma cells. BPs containing p-bromophenyl (R1 = p-Br, Ph, 2) in their side chain were the more potent to inhibit HuH7 cell viability. In addition, phenyl diesterified analogues (R2 = R3 = Ph, 2a) were more potent than methyl (R2 = R3 = Me, 2b) or non-esterified BPs (2) inducing more necrosis suggesting that they better entered into cells. Phosphodiesterase inhibitor (IBMX) reversed the effect of the esterified BPs and not that of non-esterified ones suggesting role of cell phosphodiesterases to release active BPs. BP analogues inhibited HuH7 cell migration but esterified ones had no effect on invasion due to the hiding of phosphonic groups. All together, these results indicated the therapeutic interest of these new BP prodrugs.

Induction of multiple drug resistance in HMEC-1 endothelial cells after long-term exposure to sunitinib.

Multiple drug resistance is still an unsolved problem in cancer therapy. Our previous study demonstrated that the chemotherapeutic drug doxorubicin (Dox) induced upregulation of P-glycoprotein (P-gp) in endothelial cells, resulting in a 20-fold increase in drug resistance and reduced efficiency of Dox treatment in a mice tumor model. In this study, we exposed human microvascular endothelial cells (HMEC-1) to sunitinib, a tyrosine kinase receptor inhibitor, to induce drug resistance. The results show that sunitinib treatment induced multiple drug resistance in these cells. They became resistant not only to sunitinib but also to Dox, paclitaxel, and vinblastine. Significant increases in P-gp (9.3-fold), ABCG2 (breast cancer resistance protein, 1.9-fold), and multidrug resistance-associated protein 1 (2.7-fold) gene transcription were found by quantitative polymerase chain reaction quantification, and their protein expression was confirmed by Western blot. These increases gave rise to an approximately five-fold increase in half maximal inhibitory concentration in these cells in response to sunitinib treatment in vitro. The inhibitors of adenosine triphosphate-binding cassette transporters did not reverse the drug resistance in sunitinib-resistant HMEC-1 cells, assumedly because of a blockage of the pump function caused by sunitinib. Our study indicates that the antiangiogenic drug sunitinib induces multiple drug resistance in endothelial cells. The induction of adenosine triphosphate-binding cassette transporters seems not to be responsible for observed multiple drug resistance, and the underlying mechanisms remain unknown.

ATIP3, a novel prognostic marker of breast cancer patient survival, limits cancer cell migration and slows metastatic progression by regulating microtubule dynamics.

Metastasis, a fatal complication of breast cancer, does not fully benefit from available therapies. In this study, we investigated whether ATIP3, the major product of 8p22 MTUS1 gene, may be a novel biomarker and therapeutic target for metastatic breast tumors. We show that ATIP3 is a prognostic marker for overall survival among patients with breast cancer. Notably, among metastatic tumors, low ATIP3 levels associate with decreased survival of the patients. By using a well-defined experimental mouse model of cancer metastasis, we show that ATIP3 expression delays the time-course of metastatic progression and limits the number and size of metastases in vivo. In functional studies, ATIP3 silencing increases breast cancer cell migration, whereas ATIP3 expression significantly reduces cell motility and directionality. We report here that ATIP3 is a potent microtubule-stabilizing protein whose depletion increases microtubule dynamics. Our data support the notion that by decreasing microtubule dynamics, ATIP3 controls the ability of microtubule tips to reach the cell cortex during migration, a mechanism that may account for reduced cancer cell motility and metastasis. Of interest, we identify a functional ATIP3 domain that associates with microtubules and recapitulates the effects of ATIP3 on microtubule dynamics, cell proliferation, and migration. Our study is a major step toward the development of new personalized treatments against metastatic breast tumors that have lost ATIP3 expression.

Autocrine functions of VEGF in breast tumor cells: adhesion, survival, migration and invasion.

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.

Transcriptome analysis and in vivo activity of fluvastatin versus zoledronic acid in a murine breast cancer metastasis model.

Statins and bisphosphonates are two distinct classes of isoprenoid pathway inhibitors targeting downstream enzyme to HMG-CoA reductase (upstream enzyme) and farnesyl-pyrophosphate synthase, respectively. Here, we studied fluvastatin (Fluva) and zoledronate (Zol), representative molecules of each class, respectively. In vivo metastatic potentials of both molecules were assessed. For the first time, we observed a significant reduction in progression of established metastases with Fluva treatment. Treatment with both Zol at 100 µg/kg and Fluva at 15 mg/kg inhibited 80% of the metastasis bioluminescence signal and increased survival of mice. The Zol and Fluva transcriptomic profiles of treated MDA-MB-231 cells revealed analogous patterns of affected genes, but each of them reached with different kinetics. The observable changes in gene expression started after 24 h for Fluva IC(50 72 h) and only after 48 h for Zol IC(50 72 h). To obtain early changes in gene expression of Zol-treated cells, a 3 times higher dose of Zol IC(50 72 h) had to be applied. Combining Fluva and Zol in vivo showed no synergy, but a benefit of several days in survival of mice. This study demonstrated that Zol or Fluva is of potential clinical use for the treatment of established metastasis.

Angiotensin II facilitates breast cancer cell migration and metastasis.

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.

Invading basement membrane matrix is sufficient for MDA-MB-231 breast cancer cells to develop a stable in vivo metastatic phenotype.

INTRODUCTION: The poor efficacy of various anti-cancer treatments against metastatic cells has focused attention on the role of tumor microenvironment in cancer progression. To understand the contribution of the extracellular matrix (ECM) environment to this phenomenon, we isolated ECM surrogate invading cell populations from MDA-MB-231 breast cancer cells and studied their genotype and malignant phenotype. METHODS: We isolated invasive subpopulations (INV) from non invasive populations (REF) using a 2D-Matrigel assay, a surrogate of basal membrane passage. INV and REF populations were investigated by microarray assay and for their capacities to adhere, invade and transmigrate in vitro, and to form metastases in nude mice. RESULTS: REF and INV subpopulations were stable in culture and present different transcriptome profiles. INV cells were characterized by reduced expression of cell adhesion and cell-cell junction genes (44% of down regulated genes) and by a gain in expression of anti-apoptotic and pro-angiogenic gene sets. In line with this observation, in vitro INV cells showed reduced adhesion and increased motility through endothelial monolayers and fibronectin. When injected into the circulation, INV cells induced metastases formation, and reduced injected mice survival by up to 80% as compared to REF cells. In nude mice, INV xenografts grew rapidly inducing vessel formation and displaying resistance to apoptosis. CONCLUSION: Our findings reveal that the in vitro ECM microenvironment per se was sufficient to select for tumor cells with a stable metastatic phenotype in vivo characterized by loss of adhesion molecules expression and induction of pro-angiogenic and survival factors.

A multimodal magnetic resonance imaging nanoplatform for cancer theranostics.

We describe an innovative multimodal system, which combines magnetic targeting of therapeutic agents with both magnetic resonance and fluorescence imaging into one system. This new magnetic nanoplatform consists of superparamagnetic γFe(2)O(3) nanoparticles, used clinically as an MRI contrast agent, conjugated to therapeutic molecules of the hydroxylmethylene bisphosphonate family (HMBPs): alendronate with an amine function as the terminal group. In vitro tests with breast cancer cells show that the γFe(2)O(3)@alendronate hybrid nanomaterial reduces cell viability and acts as a drug delivery system. We also investigated the anti-tumoural properties in vivo in nude mice xenografted with MDA-MB-231 tumours. We show that the presence of both γFe(2)O(3)@alendronate and a magnetic field significantly reduced the development of tumours. The amine functionalities can be used as precursor groups for the covalent coupling of peptides or monoclonal antibodies for specific biological targeting. The feasibility of this process was demonstrated by coupling rhodamine B, a fluorescence marker, to the γFe(2)O(3)@alendronate nanohybrid. The system showed fluorescent properties and high affinity for cells. Flow cytometry and fluorescence microscopy were used to study the kinetics of γFe(2)O(3)@alendronate uptake by cells. The magnetic and fluorescent nanoparticles are potential candidates for smart drug-delivery systems. Also, the superparamagnetic behaviour of such nanoparticles may be exploited as MRI contrast agents to improve therapeutic diagnostics.

New symmetrically esterified m-bromobenzyl non-aminobisphosphonates inhibited breast cancer growth and metastases.

BACKGROUND: Although there was growing evidence in the potential use of Bisphosphonates (BPs) in cancer therapy, their strong osseous affinities that contrast their poor soft tissue uptake limited their use. Here, we developed a new strategy to overcome BPs hydrophilicity by masking the phosphonic acid through organic protecting groups and introducing hydrophobic functions in the side chain. METHODOLOGY/PRINCIPAL FINDINGS: We synthesized non-nitrogen BPs (non N-BPs) containing bromobenzyl group (BP7033Br) in their side chain that were symmetrically esterified with hydrophobic 4-methoxphenyl (BP7033BrALK) and assessed their effects on breast cancer estrogen-responsive cells (T47D, MCF-7) as well as on non responsive ones (SKBR3, MDA-MB-231 and its highly metastatic derived D3H2LN subclone). BP7033Br ALK was more efficient in inhibiting tumor cell proliferation, migration and survival when compared to BP7033Br. Although both compounds inhibited tumor growth without side effects, only BP7033Br ALK abrogated tumor angiogenesis and D3H2LN cells-induced metastases formation. CONCLUSION/SIGNIFICANCE: Taken together these data suggest the potential therapeutic use of this new class of esterified Bisphosphonates (BPs) in the treatment of tumor progression and metastasis without toxic adverse effects.

A prenylation inhibitor (sodium phenylacetate) differently affects MCF-7 cell death when ras is overexpressed, partly involving P42/44, JNK and P38 kinase activations.

BACKGROUND: Sodium phenylacetate (NaPa) inhibits breast cancer cell proliferation decreasing prenylation of small G proteins including Ras. MATERIALS AND METHODS: Aponecrosis induced by NaPa in MCF-7 and MCF-7ras breast cancer cells was evaluated by measuring Annexin V/PI labelling by flow cytometry. Specific inhibitors of p42/44 (PD 98059), p38 (SB 600125) and JNK (SP 202190) in association with NaPa were also tested. Mitogen-activated kinase (MAPK) activation was measured by immunoprecipitation. RESULTS: NaPa induced cell death more efficiently (80%) in the MCF-7ras cells compared to the MCF-7 cells (60%). NaPa activated ERK 1/2 and its combination with PD 98059 decreased cell death in the MCF-7ras cells in contrast to the MCF-7 cells. Combination of NaPa with specific inhibitors of both JNK and p38 kinases also partly decreased MCF-7ras cell death. CONCLUSION: NaPa induced cell death differently when ras was overexpressed in breast cancer cells, partly involving p42/44, JNK and p38 pathways.

Distinct heparin binding sites on VEGF165 and its receptors revealed by their interaction with a non sulfated glycoaminoglycan (NaPaC).

We previously demonstrated that a non sulfated analogue of heparin, phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited angiogenesis. Here, we observed that NaPaC inhibited the VEGF165 binding to both VEGFR2 and NRP-1 and abolished VEGFR2 activity. Further, we explored the effects of NaPaC on VEGF165 interactions with its receptors, VEGFR2 and NRP-1, co-receptor of VEGFR2. Surface plasmon resonance and affinity gel electrophoresis showed that NaPaC interacted directly with VEGF165, VEGFR2 and NRP-1 but not with heparin-independent factor such as VEGF121. NaPaC completely inhibited the heparin binding to VEGF165, NRP-1 and VEGFR2. We found that NaPaC bound to all three molecules, VEGF165, VEGFR2 and NRP-1, but was more effective in inhibiting heparin binding to VEGF165. These results suggested that heparin binding sites of VEGFR2 and NRP-1 were different from those of VEGF165.