Metastasis Of Mammary Carcinoma Cells Research Articles

THU545 Growth Hormone Receptor Antagonist As A Novel Combination Therapy Against Triple Negative Breast Cancer

Abstract Disclosure: P. Mora-Criollo: None. R. Basu: None. J.J. Kopchick: None. Triple Negative breast (TNBC) cancer is classified based on the absence of ER-, PR-, and HER2- receptors and is associated with high metastatic potential. TNBC accounts for approximately 15-20% of new breast cancer diagnoses and is responsible for most breast cancer-related deaths. First-line treatment of anticancer drugs can be effective in 30-70% of patients with TNBC, but during treatment, more than half of the patients develop resistance and progress into metastatic disease. Previous studies have demonstrated the link between growth hormone receptor (GHR) expression and mammary carcinogenesis and established that GHR signaling promotes breast cancer development and progression. Conversely, inhibition of GHR expression has been shown to arrest tumor initiation and reduce metastasis of mammary carcinoma cells. Here we present the feasibility studies for employing GHR as a target for novel combination therapeutic approaches in TNBC. We performed retrospective analysis of GHR transcript expression and survival in TNBC patients, followed by experimental data in cellular models using combinations of Pegvisomant (an FDA-approved GHR antagonist) with chemotherapeutic agents, as a potential therapeutic option in TNBC. Survival probability plots from the TCGA dataset of 405 TNBC samples showed that patients expressing high levels of the GHR showed a significantly poor survival (27 months, p=0.038) compared to patients expressing low GHRs (40.37 months). Additionally, human breast cancer MDA-MB-453 cells, when treated with human (hGH)(2.5 nM) increased activation of JAK2, STAT5, SRC, ERK1/2, and AKT, associated with cancer progression. Importantly, treatment of the cells with Paclitaxel (1 nM) + (hGH)(2.5 nM) increased ABCG2 expression and the epithelial-to-mesenchymal transition (EMT) makers (SLUG, SNAIL, N-cad) associated with drug resistance and tumor progression. On the contrary, when cells were treated with Pegvisomant (500 nM) in combination with Paclitaxel + GH, a significant decrease in protein and RNA expression of ABCG2 as well as EMT markers was observed. Next, serum collected from GH antagonist (GHA) mice and WT as control, were used to treat PY8119 mouse cells in combination with Paclitaxel (1 nM) + bovine (bGH)(2.5 nM). Downregulation of protein and RNA expression levels of ABCG2 as well EMT makers in GHA serum treatment compared to WT was also found. This preliminary data shows that the GHR antagonist, Pegvisomant, in combination with anticancer drugs, can be effective in attacking drug resistance in TNBC. Key words: Pegvisomant, growth hormone receptor Presentation: Thursday, June 15, 2023

Inhibition of the invasion and metastasis of mammary carcinoma cells by NBD peptide targeting S100A4 via the suppression of the Sp1/MMP‑14 axis.

A synthetic peptide that blocks the interaction between the metastasis-enhancing calcium-binding protein, S100A4, and its effector protein, methionine aminopeptidase 2 (MetAP2) (the NBD peptide), was previously demonstrated to inhibit the angiogenesis of endothelial cells, leading to the regression of human prostate cancer in a xenograft model. However, the effects of the NBD peptide on the malignant properties of cancer cells that express S100A4 remain to be elucidated. The present study demonstrates that the NBD peptide inhibits the invasiveness and metastasis of highly metastatic human mammary carcinoma cells. The introduction of the peptide into MDA-MB-231 variant cells resulted in the suppression of matrix degradation in a gelatin invadopodia assay and invasiveness in a Matrigel invasion assay. In line with these results, the peptide significantly downregulated the expression of matrix metalloproteinase (MMP)-14 (MT1-MMP). Mechanistic analysis of the downregulation of MMP-14 revealed the suppression of the expression of the transcription factor, specificity protein 1 (Sp1), but not that of nuclear factor (NF)-κB, early growth response 1 (EGR1) or ELK3, all of which were reported to be involved in transcriptional regulation of the MMP-14 gene. At the same time, evidence suggested that the NBD peptide also suppressed Sp1 and MMP-14 expression levels in MDA-MB-468 cells. Importantly, the intravenous administration of the NBD peptide encapsulated in liposomes inhibited pulmonary metastasis from mammary gland tumors in mice with xeno-graft tumors. These results indicate that the NBD peptide can suppress malignant tumor growth through the suppression of the Sp1/MMP-14 axis. Taken together, these results reveal that the NBD peptide acts on not only endothelial cells, but also on tumor cells in an integrated manner, suggesting that the peptide may prove to be a promising cancer therapeutic peptide drug.

Jatrorrhizine inhibits mammary carcinoma cells by targeting TNIK mediated Wnt/β-catenin signalling and epithelial-mesenchymal transition (EMT)

BackgroundTraf2 and Nck interacting serine protein kinase (TNIK) is a tumour target protein which its high expression is closely related to the occurrence and development of mammary carcinoma cells. Molecular docking revealed that jatrorrhizine, a protoberberine alkaloid, exhibits good binding affinity and interaction with TNIK. However, the underlying mechanisms of jatrorrhizine targeting TNIK inhibits the proliferation and metastasis of breast cancer cells remain unclear. MethodsTo figure out the mechanisms in vitro and in vivo, the CRISPR/Cas9 technology was used to knockout TNIK gene and detected qualitatively by immunofluorescence and immunoblotting assay. The MTT cell viability assay for cytotoxicity test, the apoptosis were detected by flow cytometry, the migration and invasion were evaluated by colony formation, wound healing assay and cell invasion assay, respectively. Anticancer effects were further corroborated by 4T1/Luc homograft tumour model. ResultsThe results showed that targeted knockout of TNIK that attenuated Wnt/β-catenin signalling and epithelial-mesenchymal transition (EMT) expression, the effects were potentiated by the addition of jatrorrhizine. Moreover, jatrorrhizine distinctly inhibited the proliferation of MDA-MB-231, MCF-7 and 4T1 cells with IC50 values of 11.08 ± 1.19 μM, 17.11 ± 4.54 μM and 22.14 ± 2.87 μM, induced mitochondrial dysfunction and early apoptosis involving mitochondrial apoptotic pathway. These results were further corroborated by the 4T1 tumour-bearing mice, which showed that jatrorrhizine significantly suppressed the proliferation and metastasis of mammary carcinoma cells without obvious toxicity. ConclusionThese findings provide an overall perspective that jatrorrhizine potentially restrains TNIK regulating Wnt/β-catenin signalling and EMT expression for mammary cancer targeted therapy.

Abstract B26: Reelin signaling in tumor microenvironment is required for metastasis of mammary carcinoma cells.

Abstract We have investigated the role of reelin signaling in the microenvironment of breast cancer and found that reelin is essential for progression of mammary tumors to metastasis. Reelin signaling serves to position neurons in the developing central nervous system, but functions of this signaling pathway in other tissues have not been studied until recently. Our laboratory discovered that the reelin pathway is an important regulator of mammary gland development. We showed that reelin and Dab1, the intracellular adaptor protein for reelin, are required for timely development and proper morphology of the mammary gland. We also found that normal mammary epithelial cells (MECs) can respond to exogenous reelin by slowing their migration rate, while mouse mammary carcinoma 4T1 cells lose this response, likely through downregulation of Dab1 mRNA. Orthotopic injections of 4T1 cells into wild type Balb/C mice resulted in aggressive tumors that produced numerous metastases in the lungs by 4 weeks post-injection. However, when these cells were injected into mice that lack reelin or Dab1 (reeler and Dab1 mutant mice), we saw a dramatic reduction in lung metastases despite the presence of primary tumors comparable in size to the tumors formed in wild type mice. We show that this phenotype is dependent upon loss of reelin in the host environment, since tumor cells isolated from reeler mutant mice produce primary tumors and lung metastases when injected into Balb/C mice. Using soft agar colony assays and Western blotting, we show that the inability of 4T1 cells to metastasize in mutant animals might be due to a reduced capacity of mammary carcinoma cells to undergo epithelial to mesenchymal transition (EMT) in an environment deficient for reelin signaling. In addition, tumors from reeler and Dab1 mutant mice appear to have more differentiated cells as shown by anti-α smooth muscle Actin and anti-K14 immunostaining. Ki-67 staining showed no differences in proliferation between tumors from wild type, reeler or Dab1 mutant mice. Finally, tail vein injections of 4T1 cells into reeler mutant mice also fail to produce metastases, indicating that reelin signaling in the host environment might have important functions at several steps of the metastatic process. Our results implicate reelin signaling as an essential component of transition from the primary tumor to a metastatic carcinoma. Citation Format: Elvira Khialeeva, Joan W. Chou, Ellen M. Carpenter. Reelin signaling in tumor microenvironment is required for metastasis of mammary carcinoma cells.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B26. doi:10.1158/1538-7445.CHTME14-B26

Bone marrow-derived, alternatively activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model

IntroductionTumor-associated macrophages, which are derived from the infiltration of circulating bone marrow-derived monocytes, consist primarily of a polarized M2 macrophage (M2-Mϕ) population and are associated with poor prognosis in various cancers. In the present study, we attempted to assess whether M2-Mϕs derived from bone marrow stimulate the promotion and progression of mammary tumors.Methods4T1 murine mammary carcinoma cells were injected either alone or coupled with M2-Mϕs into the mammary fat pads of syngeneic female Balb/C mice. M2-Mϕs were prepared by treating monocytes isolated from female Balb/C mouse bone marrow with IL-4. Tumor cell growth was determined using an in vivo imaging system and the expression of cell proliferation-related, angiogenesis-related, and lymphangiogenesis-related proteins in tumor tissues was immunohistochemically analyzed. To evaluate the effects of the crosstalk between 4T1 cells and M2-Mϕs on the secretion and mRNA expression of cytokines and the migration of monocytes, 4T1 cells and M2-Mϕs were co-cultured and cytokine antibody array, real-time RT-PCR, and trans-well migration assays were conducted.ResultsThe co-injection of M2-Mϕs into the mammary fat pads of mice increased solid tumor growth and lung metastasis of 4T1 cells as well as the infiltration of CD45+ leukocytes into tumor tissues. The proportions of Ki-67+ proliferating cells and the expression of hypoxia inducible factor-1α, vascular endothelial cell growth factor A, CD31, vascular endothelial cell growth factor C, and lymphatic vessel endothelial receptor-1 were increased significantly in the tumor tissues of mice co-injected with 4T1 cells and M2-Mϕs. The in vitro results revealed that the proliferation of 4T1 cells, the migration of monocytes, and the secretion of granulocyte colony-stimulating factor, IFNγ, IL-1α, IL-2, IL-16, IFNγ-induced protein-10, keratinocyte-derived chemokine, macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and RANTES were increased when 4T1 cells were co-cultured with M2-Mϕs, as compared with when the 4T1 cells were cultured alone.ConclusionThe crosstalk between 4T1 cells and M2-Mϕs increased the production of cytokines, which may have induced immune cell infiltration into tumor tissues, tumor cell proliferation, angiogenesis, and lymph angiogenesis, thereby increasing solid tumor growth and lung metastasis.

Abstract 5562: High-fat diet feeding increases tumor growth and metastasis of mammary carcinoma cells in the 4T1 orthotopic BALB/c mouse model

Abstract We previously observed that chronic consumption of a high fat-diet (HFD) increased the breast cancer-related mortality in the 4T1 orthotopic mouse breast cancer model. In the present study, we determined whether feeding mice with a HFD for a prolonged period increases solid tumor growth and metastasis of breast cancer cells using the 4T1 orthotopic model. 4-week old, female BALB/c mice were fed a purified diet containing 60 kcal% fat (HFD) or 10 kcal% fat (control diet) for a period of 16 weeks. After 16 weeks, 4T1 mammary carcinoma cells (5 × 104 cells) were injected into the inguinal mammary fat pad of syngeneic female BALB/c mice and the mice were continuously fed the same diets. Solid tumor growth was increased in the HFD group as compared to the control diet group. Additionally, chronic consumption of the HFD markedly increased the number and volume of tumor nodules in the lung and liver. The expression of cyclin-dependent kinase (CDK)2, CDK4, cyclin D1, cyclin A, Ki67, vascular endothelial growth factor, CD31 (an angiogenesis marker), and CD45 (a monocyte/macrophage marker) was markedly increased in the tumor tissues in the HFD group as compared to control diet group. The protein levels of urokinase-type plasminogen activator (uPA), intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule-1 were significantly increased but those of plasminogen activator inhibitor-1 were decreased in the lung tissues of the HFD group as compared to control diet group. The serum levels of complement fragment 5a (C5a), interleukin (IL)-6, macrophage colony stimulating factor (M-CSF), soluble intercellular adhesion molecule (sICAM)-1, tissue inhibitors of metalloproteinase (TIMP)-1, and triggering receptor expressed on myeloid cells (TREM)-1 were up-regulated in the HFD group as compared to control diet group. We conducted in vitro assays to determine effect of these cytokines, which had been increased in the sera of mice fed on the HFD, on the cell proliferation, adhesion, and migration of 4T1 cells. sICAM-1 increased the viability of 4T1 cells. TREM-1, M-CSF, and sICAM-1 significantly increased the migration of 4T1 cells. Additionally, the capacity of 4T1 cells to adhere to strips coated with human collagen type I was increased by C5a, sICAM-1, IL-16, M-CSF, TIMP-1, and TREM-1. These results indicate that, in addition to adhesion molecules, metalloproteases and pro-angiogenic factors, the cytokines C5a, sICAM-1, IL-16, M-CSF, TIMP-1, and TREM-1 play important roles in the metastasis of mammary cancer cells in mice fed on a HFD for a prolonged period. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5562. doi:10.1158/1538-7445.AM2011-5562

Involvement of Galectin-3 with Vascular Cell Adhesion Molecule-1 in Growth Regulation of Mouse BALB/3T3 Cells

beta-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the beta-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal beta-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean beta-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.

Dietary supplementation with isolated soy protein reduces metastasis of mammary carcinoma cells in mice.

The present study investigated the effect of dietary supplementation with isolated soy protein (ISP) on pulmonary metastasis of carcinoma cells from primary mammary tumors induced by orthotopic injection of 4526 murine mammary carcinoma cells in female BALB/c mice. Three diets were compared: a basal AIN-93G diet and the basal diet supplemented with 10% or 20% ISP. After three weeks on the experimental diets, each mouse was injected 4 x 10(5) carcinoma cells into the right inguinal mammary fat pad. The primary tumors were excised when they reached a size of 1.0 cm in diameter. After surgery, mice were maintained on their respective diets for another three weeks. At necropsy, the incidence of metastasis, the number and size of macroscopic tumors, and the number of microscopic tumors in the lungs were determined. The incidence of mice with macroscopically visible tumors was 93%, 76%, and 67%, and the median number of macroscopic tumor was 5, 2, and 1 for the control, 10%, and 20% ISP groups (P < or = 0.05, 20% ISP vs. control). The median cross-sectional area of the macroscopic tumors was 0.93 mm2, 0.80 mm2, and 0.31 mm2, and the median volume was 0.73 mm3, 0.56 mm3, and 0.14 mm3 for the control, 10%, and 20% ISP groups (P < or = 0.01, 20% ISP vs. control). Histological examination revealed fewer microscopically detectable tumors in the ISP groups compared with the control. These results demonstrated that dietary supplementation with ISP reduced pulmonary metastasis of carcinoma cells from primary mammary tumors in BALB/c mice.

Lovastatin alters cytoskeleton organization and inhibits experimental metastasis of mammary carcinoma cells.

Lovastatin is a competitive inhibitor of 3-hydroxy 3-methylglutaryl coenzyme A reductase, the key regulatory enzyme of cholesterol biosynthesis. This enzyme catalyzes the formation of mevalonate, which is also the precursor of isoprenoid moieties, such as farnesol and geraniol, that are incorporated into several molecules essential for tumor cell signaling. Here, we describe that pretreatment with a non-cytotoxic concentration of lovastatin (10 microM) dramatically inhibited the metastatic ability of F311 mammary carcinoma cells in syngeneic BALB/c mice. Similarly, daily i.p. treatment of animals with a well-tolerated dose of lovastatin (10 mg/kg/day) significantly reduced the number of experimental lung metastases. In vitro, incubation of F3II monolayers in the presence of lovastatin caused a rounded-cell morphology. Immunofluorescence analysis revealed a lack of cortical actin organization, micrutubule disruption and inhibition of integrin-mediated focal contacts in lovastatin-treated cells. Exposure of F3II cells to lovastatin significantly inhibited tumor cell adhesion and migration, and coincubation with the cholesterol precursor mevalonate prevented these effects. Lovastatin reduced membrane localization of Rho protein, a signaling molecule involved in the regulation of actin-based cell motility that needs geranylation for membrane association and activation. In addition, lovastatin induced a dose-dependent inhibition in the secretion of urokinase, a key proteolytic enzyme during tumor invasion and metastasis, and a significant increase of tissue-type plasminogen activator, a marker of good prognosis in mammary cancer. These data suggest that antimetastatic properties of lovastatin are strongly associated with alterations in cytoskeleton organization and the consequent modulation of adhesion, motility and proteolysis.

Role of morphogenetic factors in metastasis of mammary carcinoma cells

We have analysed the role of the morphogenetic factors hepatocyte growth factor/scatter factor (HGF), neuregulin and E-cadherin in the process of metastasis and morphogenesis of mammary carcinoma cells. The cDNAs for HGF, neuregulin and E-cadherin were stably expressed in anaplastic human MDA MB 435 carcinoma cells. The altered cells were then injected into the mammary fat pads of nude mice, where they form tumors which can spontaneously metastasize to the lungs. We found that expression of HGF or neuregulin promoted metastasis whereas expression of the cell adhesion molecule E-cadherin was inhibitory. Moreover, expression of E-cadherin reconstituted the ability of the cells to form morphogenetic structures in matrigel cultures in response to HGF. These data demonstrate that HGF and neuregulin, which control branching or lobulo-alveolar morphogenesis of normal breast epithelium, do have metastasis-promoting effects on breast carcinoma cells. Moreover, our results suggest that the differential activities of the two factors can be explained by the degree of epithelial differentiation: induction of morphogenesis requires an intact epithelial adhesion and differentiation system, whereas induction of metastasis is observed when the cells have lost their epithelial characteristics.