Caveolin-1-mediated expression and secretion of kallikrein 6 in colon cancer cells.

Kallikreins are secreted proteases that may play a functional role and/or serve as a serum biomarker for the presence or progression of certain types of cancers. Kallikrein 6 (KLK6) has been shown to be upregulated in several types of cancers, including colon. The aims of this study were to elucidate pathways that influence KLK6 gene expression and KLK6 protein secretion in the HCT116 human colon cancer cells. Our data indicate a central role for caveolin-1 (CAV-1), the main structural protein of caveolae, in both KLK6 gene expression and protein secretion. Sucrose gradient subcellular fractionation reveals that CAV-1 and KLK6 colocalize to lipid raft domains in the plasma membrane of HCT116 cells. Furthermore, we show that CAV-1, although it does not directly interact with the KLK6 molecule, enhances KLK6 secretion from the cells. Deactivation of CAV-1, through SRC-mediated phosphorylation, decreased KLK6 secretion. We also demonstrate that, in colon cancer cells, CAV-1 increased the amount of phosphorylated AKT in cells by inhibiting the activity of the AKT-negative regulators PP1 and PP2A. This study demonstrates that proteins such as CAV-1 and AKT, which are known to be altered in colon cancer, affect KLK6 expression and KLK6 secretion

Sox2 maintains self-renewal of tumor initiating cells in osteosarcomas

Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma cell lines as well as in tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by shRNAs in independent murine osteosarcoma-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres, and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these tumor cells maintain a proliferative requirement for Sox2. Our data indicate that Sox2 is required for osteosarcoma cell self-renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway, that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self-renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas

The Effect of Fluid Flow Shear Stress and Substrate Stiffness on Yes-Associated Protein (YAP) Activity and Osteogenesis in Murine Osteosarcoma Cells

Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/ physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness—two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity—which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression

Angiotensin receptors and β-catenin regulate brain endothelial integrity in malaria.

Cerebral malaria is characterized by cytoadhesion of Plasmodium falciparum-infected red blood cells (Pf-iRBCs) to endothelial cells in the brain, disruption of the blood-brain barrier, and cerebral microhemorrhages. No available antimalarial drugs specifically target the endothelial disruptions underlying this complication, which is responsible for the majority of malaria-associated deaths. Here, we have demonstrated that ruptured Pf-iRBCs induce activation of β-catenin, leading to disruption of inter-endothelial cell junctions in human brain microvascular endothelial cells (HBMECs). Inhibition of β-catenin-induced TCF/LEF transcription in the nucleus of HBMECs prevented the disruption of endothelial junctions, confirming that β-catenin is a key mediator of P. falciparum adverse effects on endothelial integrity. Blockade of the angiotensin II type 1 receptor (AT1) or stimulation of the type 2 receptor (AT2) abrogated Pf-iRBC-induced activation of β-catenin and prevented the disruption of HBMEC monolayers. In a mouse model of cerebral malaria, modulation of angiotensin II receptors produced similar effects, leading to protection against cerebral malaria, reduced cerebral hemorrhages, and increased survival. In contrast, AT2-deficient mice were more susceptible to cerebral malaria. The interrelation of the β-catenin and the angiotensin II signaling pathways opens immediate host-targeted therapeutic possibilities for cerebral malaria and other diseases in which brain endothelial integrity is compromised

Regulation and Function of Caveolin-1 in Colorectal Carcinogenesis

Colon cancer is the second leading cause of cancer deaths in the United States of America. It is caused by the accumulation of mutations in tumor suppressors and oncogenes. The APC tumor suppressor is mutated in most diagnosed cases of colorectal cancer. Mutations in the K-RAS oncogene occur at later stages of colon cancer progression. In the present study, the transcriptional regulation of a novel target of these two genes, caveolin-1, was studied. Caveolin-1 is transcriptionally regulated by the APC tumor suppressor gene, via induction of its inducer, FOXO1 and the suppression of its transcriptional repressor, C-MYC. An activated K-RAS oncogene induces caveolin-1 transcription via activation of the P-I3 Kinase pathway. In addition to transcriptional regulation of caveolin-1, the influence of caveolin-1 expression on cellular phenotypes like signal transduction and polyamine uptake were assessed. The present studies demonstrate that caveolin-1 expression affects basal levels of AKT and ERK signaling, with an increased signaling associated with caveolin-1 expression in these colon tumor-derived cells. In addition, caveolin-1 expression positively affects signaling in response to an inflammatory stimulus like TPA. Interestingly, caveolin-1 expression leads to a decrease in the uptake of pro-tumorigenic molecules like polyamines, in the colon cell lines tested. Taken together, the data from this study suggests that caveolin-1 is transcriptionally regulated by the APC and the K-RAS gene at different stages of colorectal tumorigenesis and this in turn, leads to different phenotypes influenced by caveolin-1 expression

Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology

AbstractActivating mutations of FGFRs1–3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form

Caveolin-1 is a novel regulator of K-RAS-dependent migration in colon carcinogenesis

Caveolin-1 is an essential component of membrane caveolae. It is an important regulator of cellular processes such as signal transduction and endocytosis. We report here, for the first time, that caveolin-1 is a target of the K-RAS oncogene in colon carcinogenesis. Caveolin-1 is induced in colon cancer cells and in human colon tumor samples, in response to K-RAS activating mutations. An activated K-RAS oncogene transcriptionally induces caveolin-1 expression in human colon cancer cells and this effect is not restricted to the type of activating K-RAS mutation. Inhibition of the P-I3 Kinase-AKT pathway, but not the ERK MAPK pathway, both important K-RAS effectors, leads to a decrease in caveolin-1 expression indicating that the AKT pathway is involved in caveolin-1 expression in response to an activated K-RAS. Increased AKT signaling induces caveolin-1 expression by increasing the activity of the transcription factor, Sp1. Interestingly; caveolin-1 depletion alters K-RAS-dependent signaling by decreasing Grb2-SOS activity. Consistent with these finding, caveolin-1-depleted cells shows decreased migration in vitro. However, caveolin-1 overexpression by itself does not increase migration whereas an activated Src can increase migration in a caveolin-1-dependent manner. This increased migration is highly dependent on the RhoA GTPase, indicating that an activated K-RAS modulates migration in part via caveolin-1 induction, and increasing RhoA activity via phospho-caveolin-1. Our findings indicate that K-RAS regulates both caveolin-1 expression and other factors affecting caveolin-1 functions in colon cancer-derived cell migration. What's new? The role of the membrane protein caveolin-1 in cancer development has been a subject of controversy. Here the authors report, for the first time, that caveolin-1 is a target of the K-RAS oncogene in colon carcinogenesis. They demonstrate that caveolin-1 is induced in response to mutant K-RAS through AKT activation in colon cancer. They also identify caveolin-1 as a novel activator of K-RAS-dependent signaling and cancer cell migration. The data suggest that caveolin-1 is a conditional "oncogene" in colon cancer whose activity depends on mutant K-RAS, and that a positive feedback loop exists between K-RAS-dependent caveolin-1 expression and signaling. Copyright © 2012 UICC

SOX2 Regulates YAP1 to Maintain Stemness and Determine Cell Fate in the Osteo-Adipo Lineage

The osteoblastic and adipocytic lineages arise from mesenchymal stem cells (MSCs), but few regulators of self-renewal and early cell-fate decisions are known. Here, we show that the Hippo pathway effector YAP1 is a direct target of SOX2 and can compensate for the self-renewal defect caused by SOX2 inactivation in osteoprogenitors and MSCs. Osteogenesis is blocked by high SOX2 or YAP1, accelerated by depletion of either one, and the inhibition of osteogenesis by SOX2 requires YAP1. SOX2 favors adipogenesis and induces PPARγ, but adipogenesis can only occur with moderate levels of YAP1. YAP1 induction by SOX2 is restrained in adipogenesis, and both YAP1 overexpression and depletion inhibit the process. YAP1 binds β-catenin and directly induces the Wnt antagonist Dkk1 to dampen pro-osteogenic Wnt signals. We demonstrate a Hippo-independent regulation of YAP1 by SOX2 that cooperatively antagonizes Wnt/β-catenin signals and regulates PPARγ to determine osteogenic or adipocytic fates

Caveolin-1-Mediated Expression and Secretion of Kallikrein 6 in Colon Cancer Cells1

Kallikreins are secreted proteases that may play a functional role and/or serve as a serum biomarker for the presence or progression of certain types of cancers. Kallikrein 6 (KLK6) has been shown to be upregulated in several types of cancers, including colon. The aims of this study were to elucidate pathways that influence KLK6 gene expression and KLK6 protein secretion in the HCT116 human colon cancer cells. Our data indicate a central role for caveolin-1 (CAV-1), the main structural protein of caveolae, in both KLK6 gene expression and protein secretion. Sucrose gradient subcellular fractionation reveals that CAV-1 and KLK6 colocalize to lipid raft domains in the plasma membrane of HCT116 cells. Furthermore, we show that CAV-1, although it does not directly interact with the KLK6 molecule, enhances KLK6 secretion from the cells. Deactivation of CAV-1, through SRC-mediated phosphorylation, decreased KLK6 secretion. We also demonstrate that, in colon cancer cells, CAV-1 increased the amount of phosphorylated AKT in cells by inhibiting the activity of the AKT-negative regulators PP1 and PP2A. This study demonstrates that proteins such as CAV-1 and AKT, which are known to be altered in colon cancer, affect KLK6 expression and KLK6 secretion

Osteosarcoma cell proliferation and survival requires mGluR5 receptor activity and is blocked by Riluzole.

Osteosarcomas are malignant tumors of bone, most commonly seen in children and adolescents. Despite advances in modern medicine, the poor survival rate of metastatic osteosarcoma has not improved in two decades. In the present study we have investigated the effect of Riluzole on a human and mouse metastatic osteosarcoma cells. We show that LM7 cells secrete glutamate in the media and that mGluR5 receptors are required for the proliferation of LM7 cells. Riluzole, which is known to inhibit glutamate release, inhibits proliferation, induces apoptosis and prevents migration of LM7 cells. This is also seen with Fenobam, a specific blocker of mGluR5. We also show that Riluzole alters the phosphorylation status of AKT/P70 S6 kinase, ERK1/2 and JNK1/2. Thus Riluzole is an effective drug to inhibit proliferation and survival of osteosarcoma cells and has therapeutic potential for the treatment of osteosarcoma exhibiting autocrine glutamate signaling