Tumor suppressor miR-218 directly targets epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer, sensitizing cells to irradiation.

PURPOSE: MicroRNA-218 (miR-218) is a key regulator of numerous processes relevant to tumor progression. In the present study, we aimed to characterize the relationship between miR-218 and the Epidermal Growth Factor Receptor (EGFR) as well as to understand downstream effects in triple-negative breast cancer (TNBC). METHODS: We assessed miR-218 and EGFR expression in cell lines and publicly available primary breast cancer gene expression data. We then overexpressed miR-218 in two TNBC cell lines and investigated effects on EGFR and downstream mitogen-activated protein (MAP) kinase signaling. Luciferase reporter assay was used to characterize a direct binding interaction between miR-218 and EGFR mRNA. Digital holographic microscopy helped investigate cell migration and dry mass after miR-218 overexpression. Cell division and invasion were assessed microscopically, while radiation response after miR-218 overexpression alone or combined with additional EGFR knockdown was investigated via clonogenic assays. RESULTS: We found an inverse correlation between EGFR expression and miR-218 levels in cell lines and primary breast cancer tissues. MiR-218 overexpression resulted in a downregulation of EGFR via direct binding of the mRNA. Activation of EGFR and downstream p44/42 MAPK signaling were reduced after pre-miR-218 transfection. Cell proliferation, motility and invasiveness were inhibited whereas cell death and mitotic catastrophe were upregulated in miR-218 overexpressing cells compared to controls. MiR-218 overexpressing and EGFR siRNA-treated cells were sensitized to irradiation, more than miR-218 overexpressing cells alone. CONCLUSION: This study characterizes the antagonistic relationship between miR-218 and EGFR. It also demonstrates downstream functional effects of miR-218 overexpression, leading to anti-tumorigenic cellular changes.

Novel Mutations in ß-MYH7 Gene in Indian Patients With Dilated Cardiomyopathy.

BACKGROUND: Heart failure is a hallmark of severe hypertrophic cardiomyopathy and dilated cardiomyopathy (DCM). Several mutations in the ß-MYH7 gene lead to hypertrophic cardiomyopathy. Recently, causative mutations in the ß-MYH7 gene have also been detected in DCM from different populations. METHODS: Here, we sequenced the ß-MYH7 gene in 137 Indian DCM patients and 167 ethnically matched healthy controls to detect the frequency of mutations and their association. RESULTS: Our study revealed 27 variations, of which 7 mutations (8.0%) were detected exclusively in Indian DCM patients for the first time. These included 4 missense mutations-Arg723His, Phe510Leu, His358Leu, and Ser384Tyr (2.9%); a frameshift mutation-Asn676_T-del (1.5%); and 2 splice-site mutations (IVS17+2T) T>G and (IVS19-1G) G>A (3.6%). Remarkably, all 4 missense mutations altered evolutionarily conserved amino acids. All 4 missense mutations were predicted to be pathogenic by 2 bioinformatics tools-polymorphism phenotyping v2 (PolyPhen-2) and sorting intolerant from tolerant (SIFT). In addition, the 4 homology models of ß-MYH7-p.Leu358, p.Tyr384, p.Leu510, and p.His723-displayed root-mean-square deviations of ∼2.55 Å, ∼1.24 Å, ∼3.36 Å, and ∼3.86 Å, respectively. CONCLUSIONS: In the present study, we detected numerous novel, unique, and rare mutations in the ß-MYH7 gene exclusively in Indian DCM patients (8.0%). Here, we demonstrated how each mutant (missense) uniquely disrupts a critical network of non-bonding interactions at the mutation site (molecular level) and may contribute to development of dilated cardiomyopathy (DCM). Therefore, our findings may provide insight into the understanding of the molecular bases of disease and into diagnosis along with promoting novel therapeutic strategies (through personalized medicine).

INTRODUCTION: L'insuffisance cardiaque est une caractéristique de la cardiomyopathie hypertrophique grave et de la cardiomyopathie dilatée (CMD). Plusieurs mutations dans le gène ß-MYH7 conduisent à la cardiomyopathie hypertrophique. Récemment, les mutations causales dans le gène ß-MYH7 ont également été détectées au sein de différentes populations atteintes de CMD. MÉTHODES: Ici, nous avons séquencé le gène ß-MYH7 de 137 patients indiens atteints de CMD et de 167 témoins sains appariés selon l'origine ethnique pour détecter la fréquence des mutations et leur association. RÉSULTATS: L'étude nous a permis de révéler 27 variations, dont sept mutations (8,0 %) étaient exclusivement détectées chez les patients indiens atteints de CMD pour la première fois. Parmi ces mutations, nous avons observé quatre mutations faux-sens­Arg723His, Phe510Leu, His358Leu et Ser384Tyr (2,9 %), une mutation par déphasage­Asn676_T-del (1,5 %) et deux mutations des sites d'épissage (IVS17+2T) T>G et (IVS19-1G) G>A (3,6 %). Étonnamment, les quatre mutations faux-sens changeaient les acides aminés évolutivement conservés. Selon deux outils bioinformatiques­PolyPhen-2 (de l'anglais, polymorphism phenotyping v2) et SIFT (de l'anglais, sorting intolerant from tolerant), les quatre mutations faux-sens devaient être pathogènes. De plus, les quatre modélisations de ß-MYH7 par homologie­p.Leu358, p.Tyr384, p.Leu510 et p.His723­affichaient de façon respective des écarts quadratiques moyens de ∼2,55 Å, ∼1,24 Å, ∼3,36 Å et ∼3,86 Å. CONCLUSIONS: Dans la présente étude, nous avons détecté de nombreuses nouvelles mutations, uniques et rares, dans le gène ß-MYH7, exclusivement chez les patients indiens atteints de CMD (8,0 %). Ici, nous avons démontré comment chaque mutant (faux-sens) perturbe de manière unique un réseau essentiel d'interactions non liantes au site de mutation (moléculaire) et peut contribuer à la survenue de la CMD. Par conséquent, les conclusions de notre étude peuvent donner un aperçu des bases moléculaires de la maladie et du diagnostic tout en favorisant la promotion de nouvelles stratégies thérapeutiques (par la médecine personnalisée).

The Heparan Sulfate Sulfotransferases HS2ST1 and HS3ST2 Are Novel Regulators of Breast Cancer Stem-Cell Properties.

Heparan sulfate (HS) is a glycosaminoglycan found mainly in its protein-conjugated form at the cell surface and the extracellular matrix. Its high sulfation degree mediates functional interactions with positively charged amino acids in proteins. 2-O sulfation of iduronic acid and 3-O sulfation of glucosamine in HS are mediated by the sulfotransferases HS2ST and HS3ST, respectively, which are dysregulated in several cancers. Both sulfotransferases regulate breast cancer cell viability and invasion, but their role in cancer stem cells (CSCs) is unknown. Breast CSCs express characteristic markers such as CD44+/CD24-/low , CD133 and ALDH1 and are involved in tumor initiation, formation, and recurrence. We studied the influence of HS2ST1 and HS3ST2 overexpression on the CSC phenotype in breast cancer cell lines representative of the triple-negative (MDA-MB-231) and hormone-receptor positive subtype (MCF-7). The CD44+/CD24-/low phenotype was significantly reduced in MDA-MB-231 cells after overexpression of both enzymes, remaining unaltered in MCF-7 cells. ALDH1 activity was increased after HS2ST1 and HS3ST2 overexpression in MDA-MB-231 cells and reduced after HS2ST1 overexpression in MCF-7 cells. Colony and spheroid formation were increased after HS2ST1 and HS3ST2 overexpression in MCF-7 cells. Moreover, MDA-MB-231 cells overexpressing HS2ST1 formed more colonies and could not generate spheres. The phenotypic changes were associated with complex changes in the expression of the stemness-associated notch and Wnt-signaling pathways constituents, syndecans, heparanase and Sulf1. The results improve our understanding of breast CSC function and mark a subtype-specific impact of HS modifications on the CSC phenotype of triple-negative and hormone receptor positive breast cancer model cell lines.

HS2ST1-dependent signaling pathways determine breast cancer cell viability, matrix interactions, and invasive behavior.

Heparan sulfate proteoglycans (HSPGs) act as signaling co-receptors by interaction of their sulfated glycosaminoglycan chains with numerous signaling molecules. In breast cancer, the function of heparan sulfate 2-O-sulfotransferase (HS2ST1), the enzyme mediating 2-O-sulfation of HS, is largely unknown. Hence, a comparative study on the functional consequences of HS2ST1 overexpression and siRNA knockdown was performed in the breast cancer cell lines MCF-7 and MDA-MB-231. HS2ST1 overexpression inhibited Matrigel invasion, while its knockdown reversed the phenotype. Likewise, cell motility and adhesion to fibronectin and laminin were affected by altered HS2ST1 expression. Phosphokinase array screening revealed a general decrease in signaling via multiple pathways. Fluorescent ligand binding studies revealed altered binding of fibroblast growth factor 2 (FGF-2) to HS2ST1-expressing cells compared with control cells. HS2ST1-overexpressing cells showed reduced MAPK signaling responses to FGF-2, and altered expression of epidermal growth factor receptor (EGFR), E-cadherin, Wnt-7a, and Tcf4. The increased viability of HS2ST1-depleted cells was reduced to control levels by pharmacological MAPK pathway inhibition. Moreover, MAPK inhibitors generated a phenocopy of the HS2ST1-dependent delay in scratch wound repair. In conclusion, HS2ST1 modulation of breast cancer cell invasiveness is a compound effect of altered E-cadherin and EGFR expression, leading to altered signaling via MAPK and additional pathways.

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Anti-JAM-C therapy eliminates tumor engraftment in a xenograft model of mantle cell lymphoma.

Junctional adhesion molecule (JAM)-C is a member of the JAM family, expressed by a variety of different cell types, including human B lymphocytes and some B-cell lymphoma subtypes-in particular, mantle cell lymphoma (MCL). Treatment with anti-JAM-C pAbs reduces homing of human B cells to lymphoid organs in a NOD/SCID mouse model. In the present study, the role of JAM-C in the engraftment of human lymphoma B cells in mice was investigated. Administration of novel anti-JAM-C mAbs reduced tumor growth of JAM-C+ MCL cells in bone marrow, spleen, liver, and lymph nodes of mice. Treatment with anti-JAM-C antibodies significantly reduced the proliferation of JAM-C-expressing lymphoma B cells. Moreover, the binding of anti-JAM-C antibodies inhibited the phosphorylation of ERK1/2, without affecting other signaling pathways. The results identify for the first time the intracellular MAPK cascade as the JAM-C-driven signaling pathway in JAM-C+ B cells. Targeting JAM-C could constitute a new therapeutic strategy reducing lymphoma B-cell proliferation and their capacity to reach supportive lymphoid microenvironments.

HS3ST2 modulates breast cancer cell invasiveness via MAP kinase- and Tcf4 (Tcf7l2)-dependent regulation of protease and cadherin expression.

Heparan sulfate 3-O-sulfotransferase 2 (HS3ST2), an enzyme mediating 3-O-sulfation of heparan sulfate (HS), is silenced by hypermethylation in breast cancer. As HS has an important co-receptor function for numerous signal transduction pathways, the phenotypical changes due to HS3ST2 reexpression were investigated in vitro using high and low invasive breast cancer cell lines. Compared to controls, highly invasive HS3ST2-expressing MDA-MB-231 cells showed enhanced Matrigel invasiveness, transendothelial migration and motility. Affymetrix screening and confirmatory real-time PCR and Western blotting analysis revealed increased expression of several matrix metalloproteinases, cadherin-11, E-cadherin and CEACAM-1, while protease inhibitor and annexin A10 expression were decreased. Low invasive HS3ST2 -expressing MCF-7 cells became even less invasive, with no change in gelatinolytic MMP activity. HS3ST2 expression increased HS-dependent basal and FGF2-specific signaling through the constitutively active p44/42 MAPK pathway in MDA-MB-231 cells. Increased MAPK activation was accompanied by upregulation of ß-catenin in MDA-MB-231, and of the transcription factor Tcf4 in both cell lines. Dysregulation of Tcf4-regulated ion transporters and increased cytosolic acidification were observed in HS3ST2-expressing MDA-MB-231 cells, which is a possible underlying cause of increased chemosensitivity towards doxorubicine and paclitaxel in these cells. This study provides the first in vitro evidence of the involvement of HS3ST2 in breast cancer cell invasion and chemosensitivity.