Soluble forms of the Notch ligands Delta1 and Jagged1 promote in vivo tumorigenicity in NIH3T3 fibroblasts with distinct phenotypes.

We previously found that soluble forms of the Notch ligands Jagged1 and Delta1 induced fibroblast growth factor receptor-dependent cell transformation in NIH3T3 fibroblasts. However, the phenotypes of these lines differed, indicating distinct functional differences among these Notch ligands. In the present study, we used allografts to test the hypothesis that NIH3T3 fibroblasts that express soluble forms of Delta1 and Jagged1 accelerate tumorigenicity in vivo. With the exception of the full-length Jagged1 transfectant, all other cell lines, including the control, generated tumors when injected subcutaneously in athymic mice. Suppression of Notch signaling by the soluble ligands significantly increased tumor onset and growth, whereas full-length Jagged1 completely suppressed tumor development. In addition, there were striking differences in tumor pathology with respect to growth kinetics, vascularization, collagen content, size and number of necrotic foci, and invasiveness into the underlying tissue. Further, the production of angiogenic factors, including vascular endothelial growth factor, also differed among the tumor types. Lastly, both Jagged1- and Delta1-derived tumors contained phenotypically distinct populations of lipid-filled cells that corresponded with increased expression of adipocyte markers. The divergence of tumor phenotype may be attributed to ligand-specific alterations in Notch receptor responses in exogenous and endogenous cell populations within the allographs. Our findings demonstrate distinct functional properties for these Notch ligands in the promotion of tumorigenicity in vivo.

A novel mechanism of transcriptional repression of p27kip1 through Notch/HRT2 signaling in vascular smooth muscle cells.

Vascular smooth muscle cell (VSMC) proliferation occurs in vascular obstructive events such as atherosclerosis and restenosis. We previously showed that Notch receptors are induced in smooth muscle cells during vascular remodeling. Our goal was to determine the mechanisms employed by Notch signaling to regulate proliferation. Activation of Notch1 and Notch4 induced the VSMC-selective target genes HRT1 and HRT2, promoted cell cycle transit in smooth muscle cells, and led to loss of density-dependent growth inhibition. This was associated with a reduction in levels of the cyclin-dependent kinase inhibitor (cdk) p27(kip1). Over-expression of p27(kip1) resulted in a dose-dependent rescue of the Notch-induced phenotype and exit from the cell cycle. In addition, HRT2 expression was sufficient to promote S-phase entry, and we demonstrate that HRT2 interacts directly with the p27(kip1) promoter to repress transcription. Transcriptional repression occurred within the approximately 774 bp minimal p27(kip1) promoter region and mutational analysis demonstrated that repression is largely dependent on a conserved class-C domain. Our data show that Notch signaling acts to promote a proliferative phenotype in VSMC by modulation of the G1/S-phase checkpoint. In addition, we define a novel mechanism by which the Notch effector, HRT2, interacts directly with the class-C domain of the p27(kip1) promoter, repressing its expression. These studies identify a novel transcriptional target of HRT2, and show that Notch effectors directly control cell cycle regulatory components. We suggest that this mechanism is relevant to hyperproliferative states in VSMC seen during vascular remodeling and repair.

Mesenchymal stem cell osteodifferentiation in response to alternating electric current.

The present study addressed adult human mesenchymal stem cell (MSC) differentiation toward the osteoblastic lineage in response to alternating electric current, a biophysical stimulus. For this purpose, MSCs (chosen because of their proven capability for osteodifferentiation in the presence of select bone morphogenetic proteins) were dispersed and cultured within electric-conducting type I collagen hydrogels, in the absence of supplemented exogenous dexamethasone and/or growth factors, and were exposed to either 10 or 40 µA alternating electric current for 6 h per day. Under these conditions, MSCs expressed both early- (such as Runx-2 and osterix) and late- (specifically, osteopontin and osteocalcin) osteogenic genes as a function of level, and duration of exposure to alternating electric current. Compared to results obtained after 7 days, gene expression of osteopontin and osteocalcin (late-osteogenic genes) increased at day 14. In contrast, expression of these osteogenic markers from MSCs cultured under similar conditions and time periods, but not exposed to alternating electric current, did not increase as a function of time. Most importantly, expression of genes pertinent to the either adipogenic (specifically, Fatty Acid Binding Protein-4) or chondrogenic (specifically, type II collagen) pathways was not detected when MSCs were exposed to the aforementioned alternating electric-current conditions tested in the present study. The present research study was the first to provide evidence that alternating electric current promoted the differentiation of adult human MSCs toward the osteogenic pathway. Such an approach has the yet untapped potential to provide critically needed differentiated cell supplies for cell-based assays and/or therapies for various biomedical applications.

Reduced expression of fumarate hydratase in clear cell renal cancer mediates HIF-2α accumulation and promotes migration and invasion.

Germline mutations of FH, the gene that encodes for the tricarboxylic acid TCA (TCA) cycle enzyme fumarate hydratase, are associated with an inherited form of cancer referred to as Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). Individuals with HLRCC are predisposed to the development of highly malignant and lethal renal cell carcinoma (RCC). The mechanisms of tumorigenesis proposed have largely focused on the biochemical consequences of loss of FH enzymatic activity. While loss of the tumor suppressor gene von Hippel Lindau (VHL) is thought to be an initiating event for the majority of RCCs, a role for FH in sporadic renal cancer has not been explored. Here we report that FH mRNA and protein expression are reduced in clear cell renal cancer, the most common histologic variant of kidney cancer. Moreover, we demonstrate that reduced FH leads to the accumulation of hypoxia inducible factor- 2α (HIF-2α), a transcription factor known to promote renal carcinogenesis. Finally, we demonstrate that overexpression of FH in renal cancer cells inhibits cellular migration and invasion. These data provide novel insights into the tumor suppressor functions of FH in sporadic kidney cancer.

RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1.

Aberrant constitutive expression of NF-kappaB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERalpha)-negative breast cancers versus ERalpha-positive ones, due in part to repression of RelB synthesis by ERalpha signaling. Notably, RelB promoted a more invasive phenotype in ERalpha-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERalpha synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERalpha (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERalpha-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-kappaB subunit mediates repression, specifically of ERalpha, thereby promoting a more migratory phenotype.

Notch2 signaling induces apoptosis and inhibits human MDA-MB-231 xenograft growth.

Notch functions as an oncogene or tumor inhibitor in various cancers, and decreases in Notch2 expression are associated with increasing grade of human breast cancer. We constitutively activated Notch signaling with intracellular domain (ICD) expression in the human adenocarcinoma line MDA-MB-231. Notch2 signaling increased apoptosis, whereas Notch4ICD (int3) significantly increased cell proliferation and growth. Cells with activated Notch2 or Notch4 were injected into nu/nu mice for analysis of in vivo tumor xenograft phenotype. Tumor growth was significantly altered depending on the receptor activated. Notch2ICD potently suppressed tumor take and growth, leading to a 60% decrease in tumors and significantly smaller, necrotic tumors. Despite this, Notch2ICD tumors were highly vascularized, although the vessels were smaller and comprised a more immature network compared with Notch4ICD tumors. Notch4ICD tumors were highly aggressive and well vascularized, indicating a role for Notch4 signaling in the promotion of the malignant phenotype in addition to its transforming ability. Although both NotchICD groups expressed angiogenic factors, Notch4ICD had selective vascular endothelial growth factor-D in both tumor and host stroma, suggesting a differential regulation of cytokines that may impact vascular recruitment and autocrine tumor signaling. Our results demonstrate that Notch2 signaling is a potent inhibitory signal in human breast cancer xenografts.