4-(5-benzo(1-3)dioxol-5-yl-4-pyridin-2-yl-1h-imidazol-2-yl)benzamide and Cell-Transformation--Neoplastic

Tumors comprise cancer stem cells (CSCs) and their heterogeneous progeny within a stromal microenvironment. In response to transforming growth factor-β (TGF-β), epithelial and carcinoma cells undergo a partial or complete epithelial-mesenchymal transition (EMT), which contributes to cancer progression. This process is seen as reversible because cells revert to an epithelial phenotype upon TGF-β removal. However, we found that prolonged TGF-β exposure, mimicking the state of in vivo carcinomas, promotes stable EMT in mammary epithelial and carcinoma cells, in contrast to the reversible EMT induced by a shorter exposure. The stabilized EMT was accompanied by stably enhanced stem cell generation and anticancer drug resistance. Furthermore, prolonged TGF-β exposure enhanced mammalian target of rapamycin (mTOR) signaling. A bitopic mTOR inhibitor repressed CSC generation, anchorage independence, cell survival, and chemoresistance and efficiently inhibited tumorigenesis in mice. These results reveal a role for mTOR in the stabilization of stemness and drug resistance of breast cancer cells and position mTOR inhibition as a treatment strategy to target CSCs.

Topics: Animals; Antineoplastic Agents; Benzamides; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Dioxoles; Drug Resistance, Neoplasm; Epithelial-Mesenchymal Transition; Female; Humans; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Neoplastic Stem Cells; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Epidermal growth factor (EGF) and estrogen are potent regulators of breast tumorigenesis. Their short‑term actions on human breast epithelial cells have been investigated extensively. However, the consequence of a long‑term exposure to EGF and estrogen remains to be fully elucidated. The present study examined the effects of long‑term exposure to EGF and 17β‑estradiol on the proliferation, transformation, expression of markers of stemness, and tumorigenesis of MCF7 human breast adenocarcinoma cells. Exposure to EGF and/or 17β‑estradiol irreversibly enhanced the proliferation rate of MCF7 cells, even following withdrawal. However, in a mouse xenograft experiment, no significant difference in tumor volume was observed between tumors derived from cells exposed to EGF, 17β‑estradiol or EGF + 17β‑estradiol. Immunohistochemistry performed on tumors derived from 17β‑estradiol‑exposed cells revealed reduced cell proliferation and vessel scores, according to the results obtained using Ki67 and von Willebrand factor staining, respectively. The EGF‑ and/or 17β‑estradiol‑treated cells exhibited an increased ratio of cluster of differentiation (CD)44+/CD24‑ cells and enhanced ability to form mammospheres. Furthermore, the long‑term exposure of MCF7 cells to EGF and 17β‑estradiol altered their responsiveness to short‑term stimulatory or inhibitory treatments with EGF, 17β‑estradiol, transforming growth factor‑β1 (TGFβ1), Iressa and SB431542. Therefore, the findings indicated that sustained exposure of MCF7 cells to EGF and/or 17β‑estradiol resulted in enhanced cell proliferation and mammosphere formation, an increased ratio of CD44+/CD24‑ cells, and altered responses to short‑term treatments with EGF, 17β‑estradiol, TGFβ1, and drugs inhibiting these signaling pathways. However, this sustained exposure was not sufficient to affect tumor take or volume in a xenograft mouse model.

Topics: Animals; Benzamides; Carcinogenesis; Cell Proliferation; Cell Transformation, Neoplastic; Dioxoles; Epidermal Growth Factor; Estradiol; Female; Gefitinib; Humans; MCF-7 Cells; Mice, SCID; Models, Biological; Neoplastic Stem Cells; Phenotype; Quinazolines; Spheroids, Cellular; Tamoxifen; Xenograft Model Antitumor Assays

Human somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of key transcription factors. iPSCs have been generated from a variety of cell types. However, iPSC induction from human myoblasts has not yet been reported. Human primary skeletal myoblasts can be cultured from diagnostic muscle biopsy specimens, and thousands of lines are frozen and stored in biobanks, and are a valuable source for iPSC-based etiological and pathogenic studies. Our aim was to generate iPSCs from human skeletal myoblasts enriched from muscle biopsy samples. We used retro- or Sendai virus vector-mediated reprogramming of enriched human myoblasts from 7 donors. We show that stable iPSC lines can be generated from human myoblasts at efficiency similar to that of fibroblasts when appropriate media is used, and the efficiency of the feeder-free iPSC generation can be significantly improved by inhibitors of histone deacetylase (sodium butyrate) and TGF-β signaling (SB431542).

Topics: Adult; Animals; Antigens, Differentiation; Benzamides; Butyric Acid; Cell Culture Techniques; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Dioxoles; Female; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Induced Pluripotent Stem Cells; Infant; Infant, Newborn; Male; Mice; Mice, Nude; Middle Aged; Muscle, Skeletal; Myoblasts, Skeletal; Retroviridae; Sendai virus; Signal Transduction; Teratoma; Transduction, Genetic; Transforming Growth Factor beta; Young Adult

Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection.

Topics: Activins; Benzamides; Bone Morphogenetic Proteins; Breast Neoplasms; Calcium-Binding Proteins; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Cytoskeletal Proteins; Dioxoles; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mad2 Proteins; Nocodazole; Nuclear Proteins; Plasmids; Poly-ADP-Ribose Binding Proteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Repressor Proteins; Signal Transduction; Transfection; Transforming Growth Factor beta

Transforming growth factor beta-1 (TGF-beta) acts as both a tumour suppressor and a tumour promoter in a context-dependent manner. The tumour-promoting activities of TGF-beta are likely to result from a combination of Smad and non-Smad signalling pathways but remain poorly understood. Here we show that TGF-beta-mediated activation of RhoA is dependent on the kinase activity of ALK5 and that continuous ALK5 activity maintains basal RhoA-ROCK signalling, cell morphology and actin dynamics in serum-starved rodent fibroblasts independently of Smad2, Smad3 and Smad4. In immortalized human diploid fibroblasts, we show that oncogenic rewiring by transduction of (V12)HaRas instigates regulation of RhoA-ROCK signalling through an autocrine TGF-beta1-ALK5 pathway. Furthermore, we show that ALK5-mediated activation of RhoA is required for efficient (V12)HaRas, V-Raf and (V600E)BRAF transformation and (V12)HaRas-mediated anchorage-independent growth. These findings identify a new pro-oncogenic activity of TGF-beta and indicate that tumours harbouring (V12)HaRas and (V600E)BRAF mutations may be susceptible to TGF-beta signalling inhibitors.

Topics: Actins; Animals; Benzamides; Blotting, Western; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeleton; Dioxoles; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fluorescent Antibody Technique; Genes, ras; Guanosine Triphosphate; Humans; Mice; NIH 3T3 Cells; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins B-raf; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Smad3 Protein; Smad4 Protein; Transfection; Transforming Growth Factor beta

During the course of breast cancer progression, normally dormant tumour-promoting effects of transforming growth factor beta (TGFbeta), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro-migratory TGFbeta 'switch' in mammary epithelial cells in vitro, we found that TGFbeta stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFbeta receptor, ALK5, as evidenced by studies using short hairpin RNA-resistant ALK5 mutants in ALK5-depleted cells and in vitro kinase assays. Functionally, Smad1/5 co-depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFbeta-stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFbeta-stimulated Smad1/5 phosphorylation, which occurs through a non-canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro-migratory TGFbeta switch in mammary epithelial cells.

Topics: Activins; Animals; Benzamides; Bone Morphogenetic Proteins; Breast Neoplasms; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Dioxoles; Humans; Mice; Phosphorylation; Protein Binding; Protein Isoforms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad1 Protein; Smad5 Protein; Transforming Growth Factor beta