Mona Mostafa Mohamed

Cysteine cathepsins: multifunctional enzymes in cancer

Cysteine cathepsins are highly upregulated in a wide variety of cancers by mechanisms ranging from gene amplification to post-transcriptional modification. Their localization within intracellular lysosomes often changes during neoplastic progression, resulting in secretion of both inactive and active forms and association with binding partners on the tumour cell surface. Secreted, cell-surface and intracellular cysteine cathepsins function in proteolytic pathways that increase neoplastic progression. Direct proof for causal roles in tumour growth, migration, invasion, angiogenesis and metastasis has been shown by downregulating or ablating the expression of individual cysteine cathepsins in tumour cells and in transgenic mouse models of human cancer.

Cathepsin B: a potential prognostic marker for inflammatory breast cancer

BackgroundInflammatory breast cancer (IBC) is the most aggressive form of breast cancer. In non-IBC, the cysteine protease cathepsin B (CTSB) is known to be involved in cancer progression and invasion; however, very little is known about its role in IBC.MethodsIn this study, we enrolled 23 IBC and 27 non-IBC patients. All patient tissues used for analysis were from untreated patients. Using immunohistochemistry and immunoblotting, we assessed the levels of expression of CTSB in IBC versus non-IBC patient tissues. Previously, we found that CTSB is localized to caveolar membrane microdomains in cancer cell lines including IBC, and therefore, we also examined the expression of caveolin-1 (cav-1), a structural protein of caveolae in IBC versus non-IBC tissues. In addition, we tested the correlation between the expression of CTSB and cav-1 and the number of positive metastatic lymph nodes in both patient groups.ResultsOur results revealed that CTSB and cav-1 were overexpressed in IBC as compared to non-IBC tissues. Moreover, there was a significant positive correlation between the expression of CTSB and the number of positive metastatic lymph nodes in IBC.ConclusionsCTSB may initiate proteolytic pathways crucial for IBC invasion. Thus, our data demonstrate that CTSB may be a potential prognostic marker for lymph node metastasis in IBC.

Inhibition of cathepsin B activity attenuates extracellular matrix degradation and inflammatory breast cancer invasion.

IntroductionInflammatory breast cancer (IBC) is an aggressive, metastatic and highly angiogenic form of locally advanced breast cancer with a relatively poor three-year survival rate. Breast cancer invasion has been linked to proteolytic activity at the tumor cell surface. Here we explored a role for active cathepsin B on the cell surface in the invasiveness of IBC.MethodsWe examined expression of the cysteine protease cathepsin B and the serine protease urokinase plasminogen activator (uPA), its receptor uPAR and caveolin-1 in two IBC cell lines: SUM149 and SUM190. We utilized a live cell proteolysis assay to localize in real time the degradation of type IV collagen by IBC cells. IBC patient biopsies were examined for expression of cathepsin B and caveolin-1.ResultsBoth cell lines expressed comparable levels of cathepsin B and uPA. In contrast, levels of caveolin-1 and uPAR were greater in SUM149 cells. We observed that uPA, uPAR and enzymatically active cathepsin B were colocalized in caveolae fractions isolated from SUM149 cells. Using a live-cell proteolysis assay, we demonstrated that both IBC cell lines degrade type IV collagen. The SUM149 cells exhibit predominantly pericellular proteolysis, consistent with localization of proteolytic pathway constitutents to caveolar membrane microdomains. A functional role for cathepsin B was confirmed by the ability of CA074, a cell impermeable and highly selective cathepsin B inhibitor, to significantly reduce pericellular proteolysis and invasion by SUM149 cells. A statistically significant co-expression of cathepsin B and caveolin-1 was found in IBC patient biopsies, thus validating our in vitro data.ConclusionOur study is the first to show that the proteolytic activity of cathepsin B and its co-expression with caveolin-1 contributes to the aggressiveness of IBC.

Human monocytes augment invasiveness and proteolytic activity of inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer, and here, we examined in vitro the interactions between the human IBC cell line SUM149 and U937 human naive monocytes. We found an altered morphology, enhanced invasiveness and proteolytic activity of SUM149 cells when cultured with U937 cells or in U937-conditioned media (U937-CM). Increases in expression and activity of the cysteine protease cathepsin B and expression of caveolin-1 were also detected in SUM149 cells grown in U937-CM, thus suggesting a contribution of these proteins to the augmented invasion through and proteolysis of the extracellular matrix by the IBC cells.

Interleukin-6 Increases Expression and Secretion of Cathepsin B by Breast Tumor-Associated Monocytes

In the tumor microenvironment, monocytes respond to paracrine stimuli from breast cancer cells by secreting molecules that participate in breast cancer growth, invasion, intravasation and metastasis. Here we examined the effects of media conditioned by MDA-MB-231 human breast carcinoma cells (231-CM) on expression and secretion of proteases and secretion of cytokines by U937 human monocytes. We found that 231-CM increased U937: 1) proliferation; 2) expression, activity and secretion of the cysteine protease cathepsin B (CTSB); 3) secretion of matrix metalloproteinases (MMP)-2 and -9; and 4) secretion of interleukin-6 (IL-6) and insulin-like growth factor binding protein-1 (IGFBP-1). We further demonstrated by western blotting and enzymatic activity assays that the increases in CTSB secretion and activity induced by 231-CM could be reduced by neutralizing antibodies against IL-6. Our data suggest a role for IL-6 in increased monocyte expression and secretion of CTSB in response to soluble factors secreted by breast cancer cells.

Cytokines secreted by macrophages isolated from tumor microenvironment of inflammatory breast cancer patients possess chemotactic properties.

Although there is a growing literature describing the role of macrophages in breast cancer, the role of macrophages in inflammatory breast cancer (IBC) is unclear. The aim of present study was to isolate and characterize tumor associated macrophages of IBC and non-IBC patients and define their role in IBC. Tumor infiltrating monocytes/macrophages (CD14+ and CD68+) were measured by immunohistochemistry using specific monoclonal antibodies. Blood drained from axillary vein tributaries was collected during breast cancer surgery and the percentage of CD14+ in the total isolated leukocytes was assessed by flow cytometric analysis. CD14+ cells were separated from total leukocytes by immuno-magnetic beads technique and were cultured overnight. Media conditioned by CD14+ were collected and subjected to cytokine profiling using cytokine antibody array. Wound healing and invasion assays were used to test whether cytokines highly secreted by tumor drained macrophages induce motility and invasion of breast cancer cells. We found that macrophages highly infiltrate into carcinoma tissues of IBC patients. In addition blood collected from axillary tributaries of IBC patients is highly enriched with CD14+ cells as compared to blood collected from non-IBC patients. Cytokine profiling of CD14+ cells isolated from IBC patients revealed a significant increase in secretion of tumor necrosis factor-α; monocyte chemoattractant protein-1/CC-chemokine ligand 2; interleukin-8 and interleukin-10 as compared to CD14+ cells isolated from non-IBC patients. Tumor necrosis factor-α, interleukin-8 and interleukin-10 significantly increased motility and invasion of IBC cells in vitro. In conclusion, macrophages isolated from the tumor microenvironment of IBC patients secrete chemotactic cytokines that may augment dissemination and metastasis of IBC carcinoma cells.

Human Cytomegalovirus Infection Enhances NF-κB/p65 Signaling in Inflammatory Breast Cancer Patients

Human Cytomegalovirus (HCMV) is an endemic herpes virus that re-emerges in cancer patients enhancing oncogenic potential. Recent studies have shown that HCMV infection is associated with certain types of cancer morbidity such as glioblastoma. Although HCMV has been detected in breast cancer tissues, its role, if any, in the etiology of specific forms of breast cancer has not been investigated. In the present study we investigated the presence of HCMV infection in inflammatory breast cancer (IBC), a rapidly progressing form of breast cancer characterized by specific molecular signature. We screened for anti-CMV IgG antibodies in peripheral blood of 49 non-IBC invasive ductal carcinoma (IDC) and 28 IBC patients. In addition, we screened for HCMV-DNA in postsurgical cancer and non-cancer breast tissues of non-IBC and IBC patients. We also tested whether HCMV infection can modulate the expression and activation of transcriptional factor NF-κB/p65, a hallmark of IBC. Our results reveal that IBC patients are characterized by a statistically significant increase in HCMV IgG antibody titers compared to non-IBC patients. HCMV-DNA was significantly detected in cancer tissues than in the adjacent non-carcinoma tissues of IBC and IDC, and IBC cancer tissues were significantly more infected with HCMV-DNA compared to IDC. Further, HCMV sequence analysis detected different HCMV strains in IBC patients tissues, but not in the IDC specimens. Moreover, HCMV-infected IBC cancer tissues were found to be enhanced in NF-κB/p65 signaling compared to non-IBC patients. The present results demonstrated a correlation between HCMV infection and IBC. Etiology and causality of HCMV infection with IBC now needs to be rigorously examined.

Monocytes conditioned media stimulate fibronectin expression and spreading of inflammatory breast cancer cells in three-dimensional culture: A mechanism mediated by IL-8 signaling pathway

BackgroundInflammatory breast cancer (IBC) is the most aggressive form of breast cancer characterized by invasion of carcinoma cells into dermal lymphatic vessels where they form tumor emboli over expressing adhesion molecule E-cadherin. Although invasion and metastasis are dynamic processes controlled by complex interaction between tumor cells and microenvironment the mechanisms by which soluble mediators may regulate motility and invasion of IBC cells are poorly understood. The present study investigated the effect of media conditioned by human monocytes U937 secreted cytokines, chemokines and growth factors on the expression of adhesion molecules E-cadherin and fibronectin of human IBC cell line SUM149. Furthermore, cytokines signaling pathway involved were also identified.ResultsU937 secreted cytokines, chemokines and growth factors were characterized by cytokine antibody array. The major U937 secreted cytokines/chemokines were interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1/CCL2). When SUM149 cells were seeded in three dimensional (3D) models with media conditioned by U937 secreted cytokines, chemokines and growth factors; results showed: 1) changes in the morphology of IBC cells from epithelial to migratory spindle shape branched like structures; 2) Over-expression of adhesion molecule fibronectin and not E-cadherin. Further analysis revealed that over-expression of fibronectin may be mediated by IL-8 via PI3K/Akt signaling pathway.ConclusionThe present results suggested that cytokines secreted by human monocytes may promote chemotactic migration and spreading of IBC cell lines. Results also indicated that IL-8 the major secreted cytokine by U937 cells may play essential role in fibronectin expression by SUM149 cells via interaction with IL-8 specific receptors and stimulation of PI3K/Akt signaling pathway.

Inflammatory breast cancer: New factors contribute to disease etiology: A review

Inflammatory breast cancer (IBC) is a highly metastatic and fatal form of breast cancer. In fact, IBC is characterized by specific morphological, phenotypic, and biological properties that distinguish it from non-IBC. The aggressive behavior of IBC being more common among young women and the low survival rate alarmed researchers to explore the disease biology. Despite the basic and translational studies needed to understand IBC disease biology and identify specific biomarkers, studies are limited by few available IBC cell lines, experimental models, and paucity of patient samples. Above all, in the last decade, researchers were able to identify new factors that may play a crucial role in IBC progression. Among identified factors are cytokines, chemokines, growth factors, and proteases. In addition, viral infection was also suggested to participate in the etiology of IBC disease. In this review, we present novel factors suggested by different studies to contribute to the etiology of IBC and the proposed new therapeutic insights.

Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

BackgroundInflammatory breast cancer (IBC), a particularly aggressive form of breast cancer, is characterized by cancer stem cell (CSC) phenotype. Due to a lack of targeted therapies, the identification of molecular markers of IBC is of major importance. The heparan sulfate proteoglycan Syndecan-1 acts as a coreceptor for growth factors and chemokines, modulating inflammation, tumor progression, and cancer stemness, thus it may emerge as a molecular marker for IBC.MethodsWe characterized expression of Syndecan-1 and the CSC marker CD44, Notch-1 & -3 and EGFR in carcinoma tissues of triple negative IBC (n = 13) and non-IBC (n = 17) patients using qPCR and immunohistochemistry. Impact of siRNA-mediated Syndecan-1 knockdown on the CSC phenotype of the human triple negative IBC cell line SUM-149 and HER-2-overexpressing non-IBC SKBR3 cells employing qPCR, flow cytometry, Western blotting, secretome profiling and Notch pharmacological inhibition experiments. Data were statistically analyzed using Student’s t-test/Mann-Whitney U-test or one-way ANOVA followed by Tukey’s multiple comparison tests.ResultsOur data indicate upregulation and a significant positive correlation of Syndecan-1 with CD44 protein, and Notch-1 & -3 and EGFR mRNA in IBC vs non-IBC. ALDH1 activity and the CD44(+)CD24(-/low) subset as readout of a CSC phenotype were reduced upon Syndecan-1 knockdown. Functionally, Syndecan-1 silencing significantly reduced 3D spheroid and colony formation. Intriguingly, qPCR results indicate downregulation of the IL-6, IL-8, CCL20, gp130 and EGFR mRNA upon Syndecan-1 suppression in both cell lines. Moreover, Syndecan-1 silencing significantly downregulated Notch-1, -3, -4 and Hey-1 in SUM-149 cells, and downregulated only Notch-3 and Gli-1 mRNA in SKBR3 cells. Secretome profiling unveiled reduced IL-6, IL-8, GRO-alpha and GRO a/b/g cytokines in conditioned media of Syndecan-1 knockdown SUM-149 cells compared to controls. The constitutively activated STAT3 and NFκB, and expression of gp130, Notch-1 & -2, and EGFR proteins were suppressed upon Syndecan-1 ablation. Mechanistically, gamma-secretase inhibition experiments suggested that Syndecan-1 may regulate the expression of IL-6, IL-8, gp130, Hey-1, EGFR and p-Akt via Notch signaling.ConclusionsSyndecan-1 acts as a novel tissue biomarker and a modulator of CSC phenotype of triple negative IBC via the IL-6/STAT3, Notch and EGFR signaling pathways, thus emerging as a promising therapeutic target for IBC.