Targeting cholesterol impairs cell invasion of all breast cancer types.

BACKGROUND: Breast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth. METHODS: We used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition. RESULTS: The six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side. CONCLUSION: Together, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy.

Surface cholesterol-enriched domains specifically promote invasion of breast cancer cell lines by controlling invadopodia and extracellular matrix degradation.

Tumor cells exhibit altered cholesterol content. However, cholesterol structural subcellular distribution and implication in cancer cell invasion are poorly understood mainly due to difficulties to investigate cholesterol both quantitatively and qualitatively and to compare isogenic cell models. Here, using the MCF10A cell line series (non-tumorigenic MCF10A, pre-malignant MCF10AT and malignant MCF10CAIa cells) as a model of breast cancer progression and the highly invasive MDA-MB-231 cell line which exhibits the common TP53 mutation, we investigated if cholesterol contributes to cancer cell invasion, whether the effects are specific to cancer cells and the underlying mechanism. We found that partial membrane cholesterol depletion specifically and reversibly decreased invasion of the malignant cell lines. Those cells exhibited dorsal surface cholesterol-enriched submicrometric domains and narrow ER-plasma membrane and ER-intracellular organelles contact sites. Dorsal cholesterol-enriched domains can be endocytosed and reach the cell ventral face where they were involved in invadopodia formation and extracellular matrix degradation. In contrast, non-malignant cells showed low cell invasion, low surface cholesterol exposure and cholesterol-dependent focal adhesions. The differential cholesterol distribution and role in breast cancer cell invasion provide new clues for the understanding of the molecular events underlying cellular mechanisms in breast cancer.

Synthesis and evaluation of 2,5 and 2,6 pyridine-based CXCR4 inhibitors.

Targeting the interaction between G-Protein Coupled Receptor, CXCR4, and its natural ligand CXCL12 is a leading strategy to mitigate cancer metastasis and reduce inflammation. Several pyridine-based compounds modeled after known small molecule CXCR4 antagonists, AMD3100 and WZ811, were synthesized. Nine hit compounds were identified. These compounds showed lower binding concentrations than AMD3100 (1000nM) and six of the nine compounds had an effective concentration (EC) less than or equal to WZ811 (10nM). Two of the hit compounds (2g and 2w) inhibited invasion of metastatic cells at a higher rate than AMD3100 (62%). Compounds 2g and 2w also inhibit inflammation in the same range as WZ811 in the paw edema test at 40% reduction in inflammation. These preliminary results are the promising foundation of a new class of pyridine-based CXCR4 antagonists.

Synthesis and evaluation of 2,5-furan, 2,5-thiophene and 3,4-thiophene-based derivatives as CXCR4 inhibitors.

The interaction between G-Protein coupled receptor CXCR4 and its natural ligand CXCL12 has been linked to inflammation experienced by patients with Irritable Bowel Disease (IBD). Blocking this interaction could potentially reduce inflammatory symptoms in IBD patients. In this work, several thiophene-based and furan-based compounds modeled after AMD3100 and WZ811-two known antagonists that interrupt the CXCR4-CXCL12 interaction-were synthesized and analyzed. Fifteen hit compounds were identified; these compounds exhibited effective concentrations (EC) lower than 1000 nM (AMD3100) and inhibited invasion of metastatic cells by at least 45%. Selected compounds (2d, 2j, 8a) that inhibited metastatic invasion at a higher rate than WZ811 (62%) were submitted for a carrageenan inflammation test, where both 8a and 2j reduced inflammation in the same range as WZ811 (40%) but did not reduce inflammation more than 40%. Select compounds were also modeled in silico to show key residue interactions. These preliminary results with furan-based and thiophene-based analogues contribute to the new class on heterocyclic aromatic-based CXCR4 antagonists.

Up-regulation of Bone Morphogenetic Protein 7 by 2-Hydroxycinnamaldehyde Attenuates HNSCC Cell Invasion.

BACKGROUND/AIM: Few studies have examined the effect of 2'-hydroxycinnamaldehyde (HCA) on head and neck squamous cell carcinoma (HNSCC) cell invasion. This study examined the role of BMP7 on the anti-migration and anti-invasion activity of HCA using HNSCC cells. MATERIALS AND METHODS: Matrigel invasion and wound healing assays were conducted to investigate cell migration or invasion. BMP7 overexpression vector or siRNA mixture was used for transient regulation of gene expression. RESULTS: HCA attenuated HNSCC cell migration and spheroids Matrigel invasion without cytotoxicity. mRNA and protein expression of BMP7 increased with HCA treatment. Exogenous BMP7 overexpression without HCA treatment attenuated Matrigel invasion of cells. Furthermore, suppression of BMP7 by siRNA alleviated the inhibitory effect of HCA on the invasion of Matrigel by the cell, indicating that BMP7 is responsible for the anti-migration effect of HCA in HNSCC cells. CONCLUSION: HCA treatment led to a remarkable up-regulation of BMP7, which resulted in the attenuation of HNSCC cell invasion.

Utilizing Matrigel Transwell Invasion Assay to Detect and Enumerate Circulating Tumor Cells.

Metastasis is the cause of 90% of human cancer deaths. Circulating tumor cells (CTCs) in the peripheral blood and/or lymphatic vessels are cells shed from primary tumors and considered to be precursors of metastasis. Study of CTCs allows the serial monitoring of tumor progression and may provide predictive and prognostic biomarkers in clinic. Current CTC isolation and detection technologies encounter several challenges, including: heterogeneity of CTCs, low cell viability and/or high rate of contamination post-isolation, and the inability to distinguish viable/invasive from nonviable/nonfunctional CTCs, all of which can limit in vitro and in vivo characterization of CTCs. Here, we describe a new method to detect and enumerate of CTCs based on their invasive property.

A Specific Mixture of Nutrients Suppresses Ovarian Cancer A-2780 Tumor Incidence, Growth, and Metastasis to Lungs.

Ovarian cancer is the deadliest gynecological malignancy in women, and fifth leading cause of death. Despite advances made in chemotherapy and surgery, the average time of clinical remission is approximately 2 years and the 5-year survival rate is 45%. Thus, there is an urgent need for the development of a novel therapeutic approach to ovarian cancer treatment. We investigated the effect of a specific nutrient mixture (EPQ) containing ascorbic acid, lysine, proline, green tea extract, and quercetin on human ovarian cancer cell A-2780 in vivo and in vitro. Athymic female nude mice (n = 12) were all inoculated intraperitoneally (IP) with 2 × 106 cells in 0.1 mL of phosphate buffered saline (PBS) and randomly divided into two groups. Upon injection, the Control group (n = 6) was fed a regular diet and the EPQ group (n = 6) a regular diet supplemented with 0.5% EPQ. Four weeks later, the mice were sacrificed and tumors that developed in the ovary were excised, weighed, and processed for histology. Lungs were inspected for metastasis. In vitro, A-2780 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics. At near confluence, cells were treated with EPQ in triplicate at concentrations between 0 and 1000 µg/mL. Cell proliferation was measured via MTT assay, MMP-9 secretion via gelatinase zymography, invasion through Matrigel and morphology via hematoxylin and eosin (H & E) staining. All Control mice developed large ovarian tumors, whereas 5 out of 6 mice in the EPQ group developed no tumors, and one, a small tumor. Control mice also showed lung metastasis in 6 out of 6 mice, while no lung metastasis was evident in EPQ mice. Zymography demonstrated only MMP-9 expression, which EPQ inhibited in a dose-dependent fashion, with virtual total block at 250 µg/mL concentration. EPQ significantly inhibited invasion through Matrigel with total block at 250 µg/mL concentration. MTT showed dose-dependent inhibition of cell proliferation with EPQ, and H & E staining showed no morphological changes below 500 µg/mL EPQ. These results suggest that EPQ has therapeutic potential in the treatment of ovarian cancer by significantly suppressing ovarian tumor incidence and growth and lung metastasis, and by inhibiting MMP-9 secretion and invasion of A-2780 ovarian cancer cells.

Anticancer Efficacy of Polyphenols and Their Combinations.

Polyphenols, found abundantly in plants, display many anticarcinogenic properties including their inhibitory effects on cancer cell proliferation, tumor growth, angiogenesis, metastasis, and inflammation as well as inducing apoptosis. In addition, they can modulate immune system response and protect normal cells against free radicals damage. Most investigations on anticancer mechanisms of polyphenols were conducted with individual compounds. However, several studies, including ours, have indicated that anti-cancer efficacy and scope of action can be further enhanced by combining them synergistically with chemically similar or different compounds. While most studies investigated the anti-cancer effects of combinations of two or three compounds, we used more comprehensive mixtures of specific polyphenols and mixtures of polyphenols with vitamins, amino acids and other micronutrients. The mixture containing quercetin, curcumin, green tea, cruciferex, and resveratrol (PB) demonstrated significant inhibition of the growth of Fanconi anemia head and neck squamous cell carcinoma and dose-dependent inhibition of cell proliferation, matrix metalloproteinase (MMP)-2 and -9 secretion, cell migration and invasion through Matrigel. PB was found effective in inhibition of fibrosarcoma HT-1080 and melanoma A2058 cell proliferation, MMP-2 and -9 expression, invasion through Matrigel and inducing apoptosis, important parameters for cancer prevention. A combination of polyphenols (quercetin and green tea extract) with vitamin C, amino acids and other micronutrients (EPQ) demonstrated significant suppression of ovarian cancer ES-2 xenograft tumor growth and suppression of ovarian tumor growth and lung metastasis from IP injection of ovarian cancer A-2780 cells. The EPQ mixture without quercetin (NM) also has shown potent anticancer activity in vivo and in vitro in a few dozen cancer cell lines by inhibiting tumor growth and metastasis, MMP-2 and -9 secretion, invasion, angiogenesis, and cell growth as well as induction of apoptosis. The presence of vitamin C, amino acids and other micronutrients could enhance inhibitory effect of epigallocatechin gallate (EGCG) on secretion of MMPs. In addition, enrichment of NM with quercetin (EPQ mix) enhanced anticancer activity of NM in vivo. In conclusion, polyphenols, especially in combination with other polyphenols or micronutrients, have been shown to be effective against multiple targets in cancer development and progression, and should be considered as safe and effective approaches in cancer prevention and therapy.

Symmetrical bis-tertiary amines as novel CXCR4 inhibitors.

CXCR4 inhibitors are promising agents for the treatment of cancer metastasis and inflammation. A series of novel tertiary amine derivatives targeting CXCR4 were designed, synthesized, and evaluated. The central benzene ring linker and side chains were modified and optimized to study the structure-activity relationship. Seven compounds displayed much more potent activity than the reference drug, AMD3100, in both the binding affinity assay and the blocking of Matrigel invasion functional assay. These compounds exhibited effective concentration ranging from 1 to 100 nM in the binding affinity assay and inhibited invasion from 65.3% to 100% compared to AMD3100 at 100 nM. Compound IIn showed a 50% suppressive effect against carrageenan-induced paw inflammation in a mouse model, which was as effective as the peptidic antagonist, TN14003 (48%). These data demonstrate that symmetrical bis-tertiary amines are unique CXCR4 inhibitors with high potency.

Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.

Tumor cells acquire metastasis-associated (MA) phenotypes following genetic alterations in them which cause deregulation of different signaling pathways. Earlier, we reported that an upregulation of the Wnt-beta-catenin pathway (WP) is one of the genetic salient features of triple-negative breast cancer (TNBC), and WP signaling is associated with metastasis in TNBC. Using cBioPortal, here we found that collective % of alteration(s) in WP genes, CTNNB1, APC and DVL1 among breast-invasive-carcinomas was 21% as compared to 56% in PAM50 Basal. To understand the functional relevance of WP in the biology of heterogeneous/metastasizing TNBC cells, we undertook this comprehensive study using 15 cell lines in which we examined the role of WP in the context of integrin-dependent MA-phenotypes. Directional movement of tumor cells was observed by confocal immunofluorescence microscopy and quantitative confocal-video-microscopy while matrigel-invasion was studied by MMP7-specific casein-zymography. WntC59, XAV939, sulindac sulfide and beta-catenin siRNA (1) inhibited fibronectin-directed migration, (2) decreased podia-parameters and motility-descriptors, (3) altered filamentous-actin, (4) decreased matrigel-invasion and (5) inhibited cell proliferation as well as 3D clonogenic growth. Sulindac sulfide and beta-catenin siRNA decreased beta-catenin/active-beta-catenin and MMP7. LWnt3ACM-stimulated proliferation, clonogenicity, fibronectin-directed migration and matrigel-invasion were perturbed by WP-modulators, sulindac sulfide and GDC-0941. We studied a direct involvement of WP in metastasis by stimulating brain-metastasis-specific MDA-MB231BR cells to demonstrate that LWnt3ACM-stimulated proliferation, clonogenicity and migration were blocked following sulindac sulfide, GDC-0941 and beta-catenin knockdown. We present the first evidence showing a direct functional relationship between WP activation and integrin-dependent MA-phenotypes. By proving the functional relationship between WP activation and MA-phenotypes, our data mechanistically explains (1) why different components of WP are upregulated in TNBC, (2) how WP activation is associated with metastasis and (3) how integrin-dependent MA-phenotypes can be regulated by mitigating the WP.