Yousef G. Amaar

Insulin-like growth factor-binding protein 5 (IGFBP-5) interacts with a four and a half LIM protein 2 (FHL2)

Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells.In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies.

Effects of Secreted Frizzled-Related Protein 3 on Osteoblasts In Vitro

To examine if sFRP3s act as decoy receptors for Wnt, we examined the effects of recombinant sFRP3 on mouse osteoblast proliferation and differentiation. We found that sFRP3 unexpectedly increased osteoblast differentiation, suggesting it may act through other mechanisms besides acting as a decoy receptor for Wnts.

Ras-association domain family 1 protein, RASSF1C, is an IGFBP-5 binding partner and a potential regulator of osteoblast cell proliferation.

The goal of this study was to identify downstream signaling molecules involved in mediating the IGF‐independent effects of IGFBP‐5 in osteoblasts. We identified RASSF1C, a member of the RASSF1 gene products, as a IGFBP‐5 binding partner and as a potential mediator of IGFBP‐5 effects on ERK phosphorylation and cell proliferation.

Effect of insulin-like growth factor-1 (IGF-1) plus alendronate on bone density during puberty in IGF-1-deficient MIDI mice

Insulin-like growth factor-1 (IGF-1) increases both bone formation and bone resorption processes. To test the hypothesis that treatment with an antiresorber along with IGF-1, during the pubertal growth phase, would be more effective than IGF-1 alone to increase peak bone mass, we used an IGF-1 MIDI mouse model, which exhibits a >60% reduction in circulating IGF-1 levels. We first determined an optimal IGF-1 delivery by evaluating IGF-1 administration (2 mg/kg body weight/day) by either a single daily injection, three daily injections, or by continuous delivery via a minipump during puberty. Of the three regimens, the three daily IGF-1 injections and IGF-1 through a minipump produced a significant increase in total body bone mineral density (BMD) (6.0% and 4.4%, respectively) and in femoral BMD (4.3% and 6.2%, respectively) compared with the control group. Single subcutaneous (s.c.) administration did not increase BMD. We chose IGF-1 administration three times daily for testing the combined effects of IGF-1 and alendronate (100 microg/kg per day). The treatment of IGF-1 + alendronate for a period of 2 weeks increased total body BMD at 1 week and 3 weeks after treatment (21.1% and 20.5%, respectively) and femoral BMD by 29% at 3 weeks after treatment. These increases were significantly greater than those produced by IGF-1 alone. IGF-1, but not alendronate, increased bone length. IGF-1 and/or alendronate increased both periosteal and endosteal circumference. Combined treatment caused a greater increase in the total body bone mineral content (BMC) and periosteal circumference compared with individual treatment with IGF-1 or alendronate. Our data demonstrate that: (1) inhibition of bone turnover during puberty increases net bone density; and (2) combined treatment with IGF-1 and alendronate is more effective than IGF-1 or alendronate alone in increasing peak bone mass in an IGF-1-deficient MIDI mouse model.

Potential involvement of the interaction between insulin-like growth factor binding protein (IGFBP)-6 and LIM mineralization protein (LMP)-1 in regulating osteoblast differentiation.

Insulin‐like growth factor binding protein (IGFBP)‐6 has been reported to inhibit differentiation of myoblasts and osteoblasts. In the current study, we explored the mechanisms underlying IGFBP‐6 effects on osteoblast differentiation. During MC3T3‐E1 osteoblast differentiation, we found that IGFBP‐6 protein was down‐regulated. Overexpression of IGFBP‐6 in MC3T3‐E1 and human bone cells inhibited nodule formation, osteocalcin mRNA expression and ALP activity. Furthermore, accumulation of IGFBP‐6 in the culture media was not required for any of these effects suggesting that IGFBP‐6 suppressed osteoblast differentiation by an intracellular mechanism. A yeast two‐hybrid screen of an osteosarcoma library was conducted to identify intracellular binding partners to account for IGFBP‐6 inhibitory effects on osteoblast differentiation. LIM mineralizing protein (LMP‐1) was identified as a high affinity IGFBP‐6 binding partner. Physical interaction between IGFBP‐6 and LMP‐1 was confirmed by co‐immunoprecipitation. Fluorescent protein fusion constructs for LMP‐1 and IGFBP‐6 were transiently transfected into osteoblasts to provide evidence of subcellular locations for each protein. Coexpression of LMP‐1‐GFP and IGFBP‐6‐RFP resulted in overlapping subcellular localization of LMP‐1 and IGFBP‐6. To determine if there was a functional association of IGFBP‐6 and LMP‐1 as well as a physical association, we studied the effect of IGFBP‐6, LMP‐1 and their combination on type I procollagen promoter activity. LMP‐1 increased promoter activity while IGFBP‐6 reduced promoter activity, and coexpression of LMP‐1 with IGFBP‐6 abrogated IGFBP‐6 suppression. These studies provide evidence that overexpression of IGFBP‐6 suppresses human and murine osteoblast differentiation, that IGFBP‐6 and LMP‐1 physically interact, and supports the conclusion that this interaction may be functionally relevant. J. Cell. Biochem. 104: 1890–1905, 2008.

The RASSF1 Gene and the Opposing Effects of the RASSF1A and RASSF1C Isoforms on Cell Proliferation and Apoptosis

RASSF1A has been demonstrated to be a tumor suppressor, while RASSF1C is now emerging as a growth promoting protein in breast and lung cancer cells. To further highlight the dual functionality of the RASSF1 gene, we have compared the effects of RASSF1A and RASSF1C on cell proliferation and apoptosis in the presence of TNF-α. Overexpression of RASSF1C in breast and lung cancer cells reduced the effects of TNF-α on cell proliferation, apoptosis, and MST1/2 phosphorylation, while overexpression of RASSF1A had the opposite effect. We also assessed the expression of RASSF1A and RASSF1C in breast and lung tumor and matched normal tissues. We found that RASSF1A mRNA levels are significantly higher than RASSF1C mRNA levels in all normal breast and lung tissues examined. In addition, RASSF1A expression is significantly downregulated in 92% of breast tumors and in 53% of lung tumors. Conversely, RASSF1C was upregulated in 62% of breast tumors and in 47% of lung tumors. Together, these findings suggest that RASSF1C, unlike RASSF1A, is not a tumor suppressor but instead may play a role in stimulating survival in breast and lung cancer cells.

RASSF1C modulates the expression of a stem cell renewal gene, PIWIL1.

BackgroundRASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC).MethodsOver-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes.ResultsThe microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells.ConclusionsTaken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer.

Evidence that RASSF1C stimulation of lung cancer cell proliferation depends on IGFBP-5 and PIWIL1 expression levels.

RASSF1C is a major isoform of the RASSF1 gene, and is emerging as an oncogene. This is in contradistinction to the RASSF1A isoform, which is an established tumor suppressor. We have previously shown that RASSF1C promotes lung cancer cell proliferation and have identified RASSF1C target genes with growth promoting functions. Here, we further report that RASSF1C promotes lung cancer cell migration and enhances lung cancer cell tumor sphere formation. We also show that RASSF1C over-expression reduces the inhibitory effects of the anti-cancer agent, betulinic acid (BA), on lung cancer cell proliferation. In previous work, we demonstrated that RASSF1C up-regulates piwil1 gene expression, which is a stem cell self-renewal gene that is over-expressed in several human cancers, including lung cancer. Here, we report on the effects of BA on piwil1 gene expression. Cells treated with BA show decreased piwil1 expression. Also, interaction of IGFBP-5 with RASSF1C appears to prevent RASSF1C from up-regulating PIWIL1 protein levels. These findings suggest that IGFBP-5 may be a negative modulator of RASSF1C/ PIWIL1 growth-promoting activities. In addition, we found that inhibition of the ATM-AMPK pathway up-regulates RASSF1C gene expression.

Identification and characterization of RASSF1C piRNA target genes in lung cancer cells

RASSF1C up-regulates important genes involved in lung cancer cell growth, including a stem cell self-renewal gene, piwil1. In this article, we report the identification of small noncoding PIWI-interacting RNAs (piRNAs) in lung cancer cells over-expressing RASSF1C. A piRNA microarray screen was performed using RNA isolated from the lung cancer cell line H1299 stably over-expressing RASSF1C and corresponding control. The piRNA microarray screen identified several piRNAs that are regulated by RASSF1C and we have validated the expression of two up-regulated piRNAs (piR-34871 and piR-52200) and two down-regulated piRNAs (piR-35127 and piR-46545) in lung cancer cells with silenced and over-expressed RASSF1C using RT-PCR. We also assessed the expression of these four piRNAs in lung tumor and matched normal tissues (n = 12). We found that piR-34871 and piR-52200 were up-regulated in 58% and 50%, respectively; while piR-35127 and piR-46545 were down-regulated in 50% in lung tumor tissues tested. The expression of piR-35127 was inversely correlated with RASSF1C expression in 10/12 tumor tissues. Over-expression of piR-35127 and piR-46545 and knock-down of piR-34871 and piR-52200 significantly reduced normal lung and breast epithelial cell proliferation and cell colony formation as well as proliferation of lung cancer cell lines (A549 and H1299) and breast cancer cell lines (Hs578T and MDA-MB-231). This suggests that these novel piRNAs may potentially be involved in regulating lung cell transformation and tumorigenesis. RASSF1C may potentially modulate the expression of its piRNA target genes through attenuation of the AMPK pathway, as over-expression of RASSF1C resulted in reduction of p-AMPK, p21, and p27 protein levels.

Ras-Association Domain Family 1C Protein Enhances Breast Tumor Growth In Vivo

The Ras association domain family 1 (RASSF1) gene is a Ras effector that plays an important role in carcinogenesis. We have previously shown that silencing of RASSF1C decreases and over-expression of RASSF1C increases cell proliferation, migration, and attenuates apoptosis of breast cancer cells in vitro. To further confirm our working hypothesis that RASSF1C may play a role as a growth promoter, we have tested the growth of human breast cancer cells stably over-expressing RASSF1A or RASSF1C in nude mice. Our studies show that breast cancer cells over-expressing HA-RASSF1A developed significantly smaller tumors and cells over-expressing HA-RASSF1C developed significantly larger tumors compared to control cells expressing the vector back bone. We have confirmed the expression of HA-RASSF1A and HA-RASSF1C in tumor tissue using RT-PCR, western blotting and immunohistochemical analyses using HA-antibody. Together, our previous in vitro and current in vivo findings further support our hypothesis that RASSF1C, unlike RASSF1A, is not a tumor suppressor and rather it appears to function as tumor growth promoter in breast cancer cells.