The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis.

We previously characterized the retinoblastoma tumor suppressor protein (Rb) as a regulator of adherens junction assembly and cell-to-cell adhesion in osteoblasts. This is a novel function since Rb is predominantly known as a cell cycle repressor. Herein, we characterized the molecular mechanisms by which Rb performs this function, hypothesizing that Rb controls the activity of known regulators of adherens junction assembly. We found that Rb represses the expression of the p21-activated protein kinase (Pak1), an effector of the small Rho GTPase Rac1. Rac1 is a well-known regulator of adherens junction assembly whose increased activity in cancer is linked to perturbations of intercellular adhesion. Using nuclear run-on and luciferase reporter transcription assays, we found that Pak1 repression by Rb is transcriptional, without affecting Pak1 mRNA and protein stability. Pak1 promoter bioinformatics showed multiple E2F1 binding sites within 155 base pairs of the transcriptional start site, and a Pak1-promoter region containing these E2F sites is susceptible to transcriptional inhibition by Rb. Chromatin immunoprecipitations showed that an Rb-E2F complex binds to the region of the Pak1 promoter containing the E2F1 binding sites, suggesting that Pak1 is an E2F target and that the repressive effect of Rb on Pak1 involves blocking the trans-activating capacity of E2F. A bioinformatics analysis showed elevated Pak1 expression in several solid tumors relative to adjacent normal tissue, with both Pak1 and E2F increased relative to normal tissue in breast cancer, supporting a cancer etiology for Pak1 up-regulation. Therefore, we propose that by repressing Pak1 expression, Rb prevents Rac1 hyperactivity usually associated with cancer and related to cytoskeletal derangements that disrupt cell adhesion, consequently enhancing cancer cell migratory capacity. This de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated with cancer progression and metastasis.

The retinoblastoma protein (pRb) is a transcriptional regulator of osteoblast differentiation. Wnt signaling also regulates this process. While Wnt signaling hinders degradation of β‐catenin leading to its nuclear accumulation and transcription of Wnt target genes, lack of Wnt activity promotes β‐catenin degradation. Since inactivation of both the pRb and Wnt pathways is commonly observed in osteosarcomas, we intend to determine if these pathways are functionally linked. Using MC3T3 pRb+/+ and pRb−/− cells, we performed immunoblots of Dishevelled (Dvl), which promotes Wnt activity when phosphorylated, and qRT‐PCR analyses of c‐myc, a known Wnt target gene, and GSK3‐β, an inducer of β‐catenin degradation. We found a 2‐fold increase in the phosphorylated/total Dvl ratio in the pRb−/− relative to pRb+/+ cells. Our results also showed 50% and 80% decreases in GSK3‐β and c‐myc mRNA levels, respectively, in the pRb−/− relative to pRb+/+ cells. Our results suggest that pRb may have a dual effect on Wnt: it inhibits Wnt by inhibiting Dvl phosphorylation and increasing GSK3‐β mRNA levels, while it promotes Wnt by promoting c‐myc mRNA levels. Research supported by RCMI Grant #5G12RR003050.

A tumor suppressor commonly targeted in human cancers is the retinoblastoma (pRb) protein, a regulator of the G1/S phase transition of the cell cycle. pRb itself is inactivated with a frequency of over 90% in a subset of human tumors such as retinoblastomas, osteosarcomas, and small cell lung carcinomas. Ours studies suggest a novel role of pRb as a regulator of the activity of the small Rho/GTPase Rac1. Our data show that pRb can regulate Rac1 activity by controlling the levels of the p21-activated protein kinase 1 (PAK1), a main Rac-1 effector. qRT-PCR, immunoblot and immunofluorescence analyses showed that pRb loss in murine osteoblasts results in a dramatic increase of PAK1 mRNA and protein. In order to determine if this increase is due to increased mRNA half-we performed mRNA stability assays using culturing cells with actinomycin D to block new transcription and to assess the half-life of pre-existing PAK1 mRNA molecules. We did not find a significant change in PAK1 mRNA half-life when comparing pRb-expressing vs pRb-deficient osteoblasts, suggesting that pRb regulation of PAK1 is not post-We next tested whether PAK1 regulation by pRb occurs at the trancriptional level, a scenario that would be consistent with pRb's well characterized role as a transcriptional regulator. We used a genomic PCR strategy to amplify several fragments of the PAK1 promoter spanning an area from − 601 to + 159 downstream into the coding region. Fragments were cloned into a luciferase reporter plasmid and the resulting constructs were transfected into pRb-expressing and pRb-osteoblasts, followed by luciferase activity assays. We identified a pRb-responsive element in the promoter fragment spanning from −201 to +159 that seems to be responsible for PAK1 transcriptional repression in pRb-expressing osteoblasts, suggesting the presence of pRb-sensitive elements within this Pak1 promoter fragment. We next performed bioinformatics analyses aimed at identifying binding sites for transcription factor known for their capacity to interact with pRb. We found that this fragment of the PAK1 promoter contains binding sites for several well known transcriptional factors, notable among this being E2F transcription factors. This observation makes it tempting to speculate that pRb represses PAK1 transcription by blocking the E2F mediated transcriptional of the PAK1 gene. Taken together our results implicate pRb in the regulation of the activity of the small Rho/GTPases Rac1. This in turn brings forth the interesting possibility that pRb, acting via small Rho/GTPases, could have additional novel roles as a regulator of processes such as changes in cell shape, migration, and cytoskeletal reorganization, processes in which small Rho GTPases are known to play a role. This project is supported by PSM Institutional Funds, by the U56 Partnership between PSM and the H. Lee Moffitt Cancer Center (Pilot Grant No. 10-14352-02-03), and RISE Program 1R25GMO82406 from NIH-NIGMS. Citation Information: Cancer Res 2009;69(23 Suppl):A68.

The retinoblastoma (Rb) protein is a tumor suppressor commonly inactivated in human cancers. Our work has uncovered a role for Rb as a regulator of adherens junction assembly and cell-to-cell adhesion. This is a novel function since Rb is predominantly known as a cell cycle repressor. Our objective is to characterize the molecular mechanism by which Rb performs this function. We hypothesized that Rb controls the activity of known regulators of adherens junction assembly. Using qRT-PCR, immunoblots, and transcriptional assays with promoter-luciferase constructs, we found that Rb represses the expression of Pak1, which is a known effector of the small Rho GTPase Rac1. Notably, Rac1 is a well known regulator of adherens junction assembly whose increased activity in cancer is linked to tumor metastasis. We found that Pak1 repression by Rb is transcriptional and is E2F-dependent. Our chromatin immunoprecipitation assays showed that an Rb-E2F complex binds to an Rb-responsive element in the Pak1 promoter that is rich in E2F binding sites. This suggests that Pak1 is an E2F target and that Rb's repressive effect on Pak1 consists in blocking E2F activity. Further supporting a role for Rb in cell adhesion, microarray analyses comparing Rb-expressing with Rb-deficient cells showed that Rb transcriptionally regulates a variety of cell adhesion genes, including those coding for adherens junction cadherins. Pak1 also appeared among the transcript repressed by Rb, validating Rac1 and Pak1 as links between Rb and adherens junctions. Importantly, we have evidence suggesting that the de-regulation in cell adhesion-related gene expression due to Rb loss may be related to lung cancer development. We examined the Director's Challenge lung cancer database and found that numerous Rb-regulated cell adhesion genes correlate with overall survival in lung adenocarcinoma patients. For this analysis we arbitrarily defined an RB1 signature as all RB1-regulated genes with >2 activation or repression with statistically significant p-values. We found that a total of 1,154 RB1-regulated genes correlated with overall survival. This analysis suggests that a subset of RB1-regulated cell adhesion genes may be critical in the development of lung cancer. Three examples of RB1-activated cell adhesion genes that correlate with overall survival are E cadherin (Cdh 1), Integrin alpha 1 (Itga1) and Integrin alpha 10 (ItgA10). In summary, three main conclusions emerge from our studies. First, Rb transcriptionally controls expression of cadherins and other adherens junction-related genes. Second, Rb also controls adherens junction assembly by repressing Rac1 and its effector Pak1. Third, global de-regulation of cell adhesion due to Rb loss could be part of the molecular events associated to lung cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4025. doi:10.1158/1538-7445.AM2011-4025